UNIVERSITY  OF  CALIFORNIA 

MEDICAL  CENTER  LIBRARY 

SAN  FRANCISCO 


WORKS  BY 
CHARLES    F.    BOLDUAN,    M.D. 

PUBLISHED  BY 

JOHN  WILEY  &  SONS 

Immune  Sera. 

Antitoxins,  Agglutinins,  Haemolysins,  Bacterio- 
lysins,  Precipitins,  Cytotoxins,  and  Opsonins. 
New  edition,  rewritten.  By  Charles  F.  Bolduan, 
M.D.  12mo,viii+ 176  pages.  Cloth,  $1.50. 

TRANSLATIONS. 
The  Suppression  of  Tuberculosis. 

Together  with  Observations  concerning  Phthisio- 
genesis  in  Man  and  Animals,  and  Suggestions  con- 
cerning the  Hygiene  of  Cow  Stables  and  the  Pro- 
duction of  Milk  for  Infant  Feeding,  with  Special 
Reference  to  Tuberculosis.  By  Professor  E.  von 
Behring,  University  of  Marburg.  Authorized 
Translation  by  Charles  F.  Bolduan,  M.D.  12mo, 
vi  +  85  pages.  Cloth,  $1.00. 

Manual  of  Serum  Diagnosis. 

By  Doctor  O.  Rostoski,  University  of  Wurzburg. 
Authorized  Translation  by  Charles  F.  Bolduan, 
M.D.  12mo,  vi  + 86  pages.  Cloth,  $1.00. 

Studies  in  Immunity. 

By  Professor  Paul  Ehrlich.  Translated  by  Charles 
F.  Bolduan,  M.D.  Second  Edition,  Revised  and 
Enlarged.  8vo,  xi  + 712  pages.  Cloth,  $6.00. 


STUDIES  IN  IMMUNITY 


BY 


PROFESSOR   PAUL/^EHRLICH 

PRIVY  COUNCILOR  AND  DIRECTOR  OP  THE  ROYAL  INSTITUTE  FOR  EXPERIMENTAL  THERAPY, 

FRANKFURT,  GERMANY 


AND     HIS     COLLABORATORS 


COLLECTED    AXD    TRANSLATED 

BY 

DR.  CHARLES  BOLDUAN 

BACTERIOLOGIST.  RESEARCH  LABORATORY,  DEPARTMENT  OF  HEALTH,  CITY  OP  NEW  YORK 


SECOND    EDITION,    REVISED    AND    ENLARGED 


?l 

NEW  YORK 

JOHN  WILEY  &    SONS 

LONDON:    CHAPMAN  &  HALL,    LIMITED 

1910 


Copyright,  1906,  1910 

BY 

CHARLES  BOLDUAN 


Scientific  $«ss 

anb  Company 
fork 


To 
DR.     A  L  T  H  O  F  F 

PRIVY  COUNCILOR,  DIRECTOR  IN  THE  PRUSSIAN  MINISTRY  OF  EDUCATION,  BERLIN,  ETC, 

THE  ABLE  FRIEND  AND  PROMOTER  OF  MEDICAL  SCIENCE 

THIS  VOLUME  is  DEDICATED  IN 

GRATEFUL  APPRECIATION 


TRANSLATOR'S    PREFACE  TO  THE  FIRST  EDITION. 


No  apology  is  needed  for  presenting  this  translation  of  Ehrlich's 
classic  studies  in  immunity,  for  a  thorough  knowledge  of  the  master's 
work  is  indispensable  to  all  workers  in  this  field. 

Attention  is  called  to  the  fact  that  the  important  work  done  since 
the  publication  of  the  German  edition  has  been  included  by  the 
addition  of  three  chapters,  two  by  Ehrlich  and  Sachs  and  one,  written 
expressly  for  this  translation,  by  Prof.  Ehrlich.  The  subject  is  thus 
brought  up  to  about  March,  1906. 

CHARLES  BOLDUAN. 

PREFACE  TO  THE  SECOND  EDITION. 


THE  exhaustion  of  the  first  edition  of  this  work  affords  the 
translator  the  welcome  opportunity  to  add,  not  only  Professor 
Ehrlich's  new  studies,  but  also  some  of  his  earlier  papers  which 
the  recent  publication  of  Bordet's  Studies  on  Immunity  renders 
desirable. 

The  translator  may  be  pardoned  for  a  feeling  of  gratification 
at  the  cordial  reception  extended  this  book  both  by  the  medical 
press  and  the  profession  at  large.  The  appreciation  shown  has 
made  the  arduous,  and  usually  thankless,  work  of  translation  one 
of  great  pleasure. 

An  exhaustive  index  has  been  added  to  this  edition  and  will, 
it  is  hoped,  greatly  enhance  its  value  as  a  work  of  reference. 

For  kind  permission  to  reproduce  articles  from  their  publica- 
tions, thanks  are  due  to  Messrs.  August  Hirschwald,  Georg  Thieme, 
J.  F.  Lehmann,  and  Gustav  Fischer. 

CHARLES  BOLDUAX. 
NEW  YORK,  February  1,  1910. 

Hi 


PREFACE  TO  THE  GERMAN  EDITION. 


THE  present  volume  embraces  the  greater  portion  of  the  studies 
in  immunity  published  during  the  past  few  years  by  myself  and  my 
collaborators.  While  the  publication  of  these  studies  in  a  single 
volume  meets  the  request  of  numerous  workers  in  immunity,  it  is 
hoped  that  the  collection  will  at  the  same  time  fulfill  another  purpose, 
namely,  to  show  clearly  that  my  theory  of  immunity  rests  on  so 
broad  an  experimental  basis  that  it  is  practically  identical  with  a 
summary  of  generalizations  derived  from  an  enormous  mass  of  experi- 
mental data.1 

When  Behring's  great  discovery  of  antitoxin  opened  new  paths 
for  the  study  of  immunity  it  was  at  once  clear  that  further  progress 
could  be  attempted  in  two  ways.  The  first  of  these,  having  practical 
therapeutic  results  in  mind,  consists  in  bending  all  efforts  to  the  pro- 
duction of  various  individual  curative  sera.  The  other  method  con- 
sists in  seeking  a  deeper  insight  into  the  nature  of  immunity  phe- 
nomena, and  discovering  the  general  principles  underlying  the  same, 
for  these  in  turn  will  aid  practical  progress. 

By  pursuing  the  latter  method  it  has  been  found  that  the  immunity 
reaction  is  merely  a  repetition  of  certain  processes  of  normal  meta- 
bolism, and  that  what  is  apparently  a  wonderful  adaptation  to  the 
purpose  is  nothing  more  than  the  ever-recurring  manifestation  of 
primeval  wisdom  inherent  in  the  protoplasm.  I  have  endeavored  to 
establish  this  experimentally  and  to  show  that  the  bond  between 

1  With  a  view  of  giving  the  reader  a  better  idea  of  the  technique  ordinarily 
employed,  and  thereby  to  facilitate  his  introduction  to  this  subject,  I  have  had 
my  colleagues,  Dr.  Morgenroth  and  Prof.  Neisser,  present  the  result  of  their  ex- 
tensive technical  experiences  with  hsemolytic  and  bacteriolytic  test-tube  experi- 
ments, in  two  special  chapters.  (Chapters  XXIX  and  XXX.) 

v 


vi  PREFACE  TO  THE  GERMAN   EDITION. 

what  are  at  first  sight  very  dissimilar  biological  processes  is  really 
a  conception  of  the  simplest  kind. 

The  toxic  metabolic  products  of  bacteria,  the  artificially  produced 
bacteriolysins,  haemolysins,  and  cytotoxins,  and  the  majority  of  the 
ferments,  probably  always  produce  their  effects  by  the  co-action  of 
two  active  groups  in  the  molecule.  One  of  these  effects  the  union 
with  the  substance  to  be  acted  upon,  while  the  other  really  produces 
the  characteristic  effect. 

It  is  not  surprising,  in  view  of  the  enormous  multiplicity  of  the 
vital  phenomena,  that  this  simple  principle  exhibits  the  greatest 
variations  in  individual  cases.  Certainly  this  corresponds  entirely 
to  what  we  constantly  observe  in  the  domain  of  biology.  The  cell, 
for  example,  occurs  as  a  type  in  every  living  form,  from  the  lowest 
plant  to  the  highest  animal.  In  principle  it  is  ever  the  same;  in  the 
details  of  its  structure,  however,  it  is  of  endless  variety. 

But  even  from  such  complex  phenomena  as  are  exhibited,  for 
example,  by  the  artificially  produced  hsemolysins,  it  is  possible  to 
develop  the  fundamental  principles  of  my  theory,  and  thereby  give 
a  harmonious  uniform  explanation  of  the  manifold  phenomena  with 
their  peculiar  specific  relations. 

My  theory  has  developed  essentially  on  the  basis  of  chemical 
conceptions.  I  have  been  more  and  more  forcibly  impressed  with 
the  idea  that  in  a  study  of  the  fundamental  biological  phenomena, 
the  significance  of  morphological  structure  is  far  less  than  the  sig- 
nificance of  the  chemistry  involved.  It  is  obvious  that  in  order  to 
effect  a  given  chemical  process  certain  mechanical  conditions  must 
be  fulfilled.  In  other  words  the  production  of  any  chemical  action 
necessitates  the  presence  and  the  suitable  arrangement  of  apparatus. 
The  essential  feature,  however,  is  neither  apparatus  nor  form,  but 
the  constituents  involved;  for  without  changing  the  apparatus 
hundreds  of  different  combinations  can  be  effected  according  to  the 
components  employed.  Similarly  in  biology  I  believe  that  the  morpho- 
logical arrangement  of  the  organs  and  cells  is  not  the  essential  feature, 
but  that  this  is  rather  to  be  sought  for  in  chemical  differences  of  the 
constituents. 

I  am  convinced  that  the  influence  exerted  by  my  theory  will 
extend  far  beyond  the  limits  of  pure  immunity  studies,  and  that  it 
is  of  considerable  significance  for  an  appreciation  of  vital  phenomena. 
Furthermore,  I  believe  that  the  theory  is  of  great  value  in  studying 
certain  phenomena  which  dominate  all  life,  namely,  intracellular 


PREFACE  TO  THE  GERMAN  EDITION.  vii 

metabolism,  especially  its  two  main  phases,  anabolism  and  catabolism^ 
It  has  been  shown  that  the  substances  obtained  by  immunization 
are  nothing  but  the  tools  of  normal  cell-life,  tools  which  we  can  thus 
isolate  from  their  place  of  production  and  subject  to  an  individual 
examination.  This  at  once  opens  new  paths  for  approaching  the 
study  of  vital  phenomena,  which  embraces  not  only  the  physiology 
and  pathology  of  metabolism,  but  also  certain  other  physiological 
problems  such  as  those  of  secretion,  heredity,  etc. 


At  the  recent  Congress  for  Hygiene  and  Demography  (Brussels), 
in  which  the  chief  problems  of  immunity  were  discussed,  it  was  seen 
that  my  theory  is  not  yet  accepted  by  all  the  workers  in  this  subject, 
there  being  still  a  few  opponents.  This  was  to  be  expected.  Cer- 
tainly nothing  is  more  desirable  in  all  scientific  problems  than  the 
expression  of  different  opinions,  for  as  a  result  of  experimental  studies 
they  lead  to  a  deeper  insight  into  the  subject  in  question.  Hence 
it  is  largely  the  opposition  of  Bordet  and  other  distinguished 
workers  in  the  Pasteur  Institute  that  has  spurred  us  on  in  our  experi- 
mental labors,  and  caused  us  to  establish  the  amboceptor  theory 
more  firmly  than  ever. 

On  the  other  hand  it  is  very  annoying  when  such  authors  as  Gruber, 
who  have  absolutely  no  personal  experience  in  the  main  questions, 
wage  a  bitter  war  merely  because  they  have  made  a  few  literary 
studies;  it  is  the  more  exasperating  since  they  seek  to  make  up  the 
deficiencies  in  their  arguments  by  the  intensity  and  personality 
of  their  attacks.  Such  authors  are  in  no  position  to  correctly  orientate 
themselves  in  the  mass  of  true  and  false  observations  that  each  day's 
literature  brings  forth. 

It  was  a  great  pleasure,  therefore,  to  see  one  of  the  founders  of 
the  doctrine  of  immunity,  R.  Pfeiffer,  and  that  distinguished  repre- 
sentative of  Paltauf's  Institute  in  Vienna,  R.  Kraus,  express  them- 
selves in  favor  of  my  theory.  They  confessed  they  had  both  really 
opposed  the  theory  from  the  start,  and  that  the  main  purpose  in  devis- 
ing their  various  experiments  had  been  to  show  that  it  was  untenable. 
Just  these,  however,  had  convinced  them  that  the  side-chain  theory 
not  only  afforded  the  best  explanation  for  their  results,  but  had  even 
enabled  them  to  predict  these  results.  The  chief  problems  now 
under  discussion  are  :  (1)  the  constitution  of  active  cytotoxic  sub- 


viii  PREFACE  TO  THE  GERMAN  EDITION. 

stances,  whether  or  not  they  are  made  up  of  two  parts  possessing 
different  functions;  (2)  the  union  of  specific  amboceptors  with  the 
complements;  (3)  the  plurality  of  complements.  I  am  convinced 
that  the  near  future  will  furnish  so  many  additional  arguments  for 
the  correctness  of  my  views  that  all  of  these  questions,  as  well  as 
numerous  others,  will  be  decided  in  my  favor.  And  the  decision,  I 
believe,  will  not  be  merely  in  favor  of  my  views  in  general,  but  will 
extend  even  to  the  details. 

In  a  way,  therefore,  my  position  is  like  that  of  a  chess-player  who. 
even  though  his  game  is  won,  is  forced  by  the  obstinacy  of  his  opponent 
to  carry  it  on  move  by  move  until  the  final  "mate.'' 


For  the  means  to  carry  on  these  experiments,  I  am  indebted  first 
of  all  to  the  intelligent  support  which  my  scientific  aims  have  received 
at  the  hands  of  my  superiors,  the  Prussian  Ministry  of  Education. 
I  am  especially  grateful  to  the  ministerial  director,  Dr.  Althoff,  who 
aided  me  in  every  way  possible,  and  exerted  himself  to  lighten  my 
scientific  labors.  I  may  say  that  I  was  first  spurred  on  to  the  im- 
munity studies  contained  in  "Die  Werthbemessung  des  Diphtherie- 
heilserums/'  and  which  Lhave  led  to  the  formulation  of  the  side-chain 
theory,  by  the  remarks  addressed  to  me  by  Dr.  Althoff  when  the 
Institute  was  founded.  It  was  he  who  begged  that  my  first  problem 
be  an  exhaustive  study  whereby  the  difficulties  which  had  arisen 
in  titrating  and  standardizing  diphtheria  antitoxin  might  be  overcome. 
To  this  kind  and  able  friend  I  have  therefore  dedicated  this  volume  as 
a  token  of  my  gratitude  and  esteem. 

PAUL  EHKLICH. 
FRANKFURT  A.  M.,  February  1904. 


CONTENTS. 


(CHAPTEB  PAGB 

I.  CONTRIBUTIONS  TO  THE  THEORY  OF 

LYSIN  ACTION Ehrlich  and  Morgenroth.       1 

II.  CONCERNING  H^MOLYSINS.  (Second 

Communication.) Ehrlich  and  Morgenroth.     11 

III.  STUDIES   ON   H^MOLYSINS.     (Third 

Communication.) Ehrlich  and  Morgenroth.     23 

IV.  CONTRIBUTIONS  TO  THE  STUD     OF 

IMMUNITY von  Dungem.     36 

New  Experiments  on  the  Side- 
chain  Theory.     Phagocytosis  and 
Glohulicidal  Immuity. 
V.  CONTRIBUTIONS  TO  THE  STUDY  OF 

IMMUNITY von  Dungern.     47 

Receptors  and  the  Formation  of 

Antibodies.    Milk  Immune  Serum. 

VI.  STUDIES  ON  H^MOLYSINS.     (Fourth 

Communication.) Ehrlich  and  Morgenroth.     56 

VII.  STUDIES    ON    H^EMOLYSINS.     (Fifth 

Communication.) Ehrlich  and  Morgenroth.     71 

VIII.  STUDIES   ON    HJSMOLYSINS.     (Sixth 

Communication.) Ehrlich  and  Morgenroth.     88 

IX.  CONCERNING  THE  MODE  OF  ACTION 

OF  BACTERICIDAL  SERA M.  Neisser.  120 

X.  THE  DEFLECTION  OF  COMPLEMENTS 
IN  BACTERICIDAL  TEST-TUBE  EX- 
PERIMENTS  Lipstein.  132 

XI.  ACTIVE   IMMUNITY    AND   OVERNEU- 

TRALIZED  DIPHTHERIA  TOXINS Rehns.  143 

XII.  Is  IT  POSSIBLE  BY  INJECTING  AG- 
GLUTINATED TYPHOID  BACILLI  TO 
CAUSE  THE  PRODUCTION  OF  AN 

AGGLUTININ? M.  Neisser.  146 

XIII.  IMMUNIZING  EXPERIMENTS  WITH 
ERYTHROCYTES  LADEN  win  IM- 
MUNE BODY Sachs.  158 

ix 


;  CONTENTS. 

CHAPTER  PAGE 

XIV.  THE  ESCAPE  OF  HEMOGLOBIN  FROM 
BLOOD-CELLS     HARDENED     WITH 

CORROSIVE  SUBLIMATE Sachs.  163 

XV.  A  CONTRIBUTION  TO  THE  STUDY  OF 

THE      POISON      OF      THE      COMMON 

GARDEN  SPIDER Sachs.  167 

XVI.  A  STUDY  OF  TOAD  POISON Proscher.  175 

XVII.  CONCERNING  ALEXIN  ACTION , Sachs.  181 

XVIII.  CONCERNING    THE     PLURALITY    OF 

COMPLEMENTS  OF  THE  SERUM Ehrlich  and  Sachs.  195 

XIX.  CONCERNING    THE    MECHANISM    OF 

THE  ACTION  OF  AMBOCEPTORS Ehrlich  and  Sachs.  209 

XX.  DIFFERENTIATING  COMPLEMENTS  BY 
MEANS  OF  A   PARTIAL  ANTICOM- 

PLEMENT Marshall  and  Morgenroth.  222 

XXI.  CONCERNING     THE     COMPLEMENTO- 

PHILE      GROUPS      OF      THE      AMBO- 
CEPTORS  Ehrlich  and  Marshall.   226 

XXII.  CONCERNING     THE     COMPLEMENTI- 

BILITY  OF  THE  AMBOCEPTORS Morgenroth  and  Sachs.  233 

XXIII.  THE  PRODUCTION  OF  HEMOLYTIC 
AMBOCEPTORS  BY  MEANS  OF 

SERUM  INJECTIONS Morgenroth.  241 

XXIV.  THE  QUANTITATIVE  RELATIONS  BE- 
TWEEN AMBOCEPTOR,  COMPLE- 
MENT, AND  ANTICOMPLEMENT Morgenroth  and  Sachs.  250 

XXV.  THE    HEMOLYTIC    PROPERTIES    OF 

ORGAN  EXTRACTS Korschun  and  Morgenroth.  267 

XXVI.  REVIEW  OF  BESREDKA'S  STUDY, 
"  LES  ANTIHEMOLYSINES  NATU- 

RELLES  " Marshall  and  Morgenroth.  283 

XXVII.  THE   MODE   OF   ACTION   OF  COBRA 

VENOM Kyes.  291 

XXVIII.  FURTHER  STUDIES  ON  THE  DYSEN- 
TERY BACILLUS Shiga.  312 

XXIX.  METHODS    OF    STUDYING    H.EMOLY- 

SINS Morgenroth.  326 

XXX.  THE  TECHNIQUE  OF  BACTERICIDAL 

TEST-TUBE  EXPERIMENTS M .  Neisser.  348 

XXXI.  THE  PROPERTY  OF  THE  BRAIN  TO 

NEUTRALIZE  TETANUS  TOXIN Marx.  356 

XXXII.  THE    PROTECTIVE    SUBSTANCES    OF 

THE  BLOOD Ehrlich.  364 

XXXIII.  THE  RECEPTOR  APPARATUS  OF  THE 

RED  BLOOD-CELLS  . . ,  . , Ehrlich.  390 


CONTENTS  XI 

CHAPTER  PAGE 

XXXIV.  THE  RELATIONS  EXISTING  BETWEEN 
CHEMICAL  CONSTITUTION,  DISTRI- 
BUTION, AND  PHARMACOLOGICAL 

ACTION Ehrlich.  404 

XXXV.  A  STUDY  OF  THE  SUBSTANCES  WHICH 

ACTIVATE  COBRA  VENOM Kyes  and  Sachs  443 

XXXVI.  THE    ISOLATION    OF    SNAKE-VENOM 

LECITHIDS Kyes.  466 

XXXVII.  THE  CONSTITUENTS  OF  DIPHTHERIA 

TOXIN Ehrlich  481 

XXXVIII.  TOXIN  AND  ANTITOXIN:   A  Reply  to 

the  Latest  Attack  of  Gruber Ehrlich.  514 

XXXIX.  THE  RELATIONS  EXISTING  BETWEEN 

TOXIN  AND  ANTITOXIN  AND  THE 

METHODS  OF  THEIR  STUDY Ehrlich  and  Sachs.  547 

XL.  THE  MECHANISM  OF  THE  ACTION  OF 

ANTIAMBOCEPTORS Ehrlich  and  Sachs.  561 

XLI.  A  GENERAL  REVIEW  OF  THE  RECENT 

WORK  IN  IMMUNITY Ehrlich.  577 

XLII.  THE  MULTIPLICITY  OF  ANTIBODIES 

OCCURRING  IN  NORMAL  SERUM Neisser.  587 

XLIII.  THE  BINDING  OF  H^EMOLYTIC  AMBO- 

CEPTORS Morgenroth.  595 

XLIV.  THE  JOINT  ACTION  OF  NORMAL  AND 
IMMUNE  AMBOCEPTORS  IN  HAE- 
MOLYSIS   Sachs.  601 

XLV.  THE  POWER  OF  NORMAL  SERUM  TO 

DEFLECT  COMPLEMENT Sachs.  610 

XLVI.  THE  JOINT  ACTION  OF  SEVERAL  AM- 
BOCEPTORS IN  HAEMOLYSIS  AND 

THEIR  RELATION  TO  THE  COMPLE- 
MENTS   Sachs  and  Bauer.  616 

XLVII.  STUDIES  IN  AMBOCEPTORS Browning  and  Sachs.  649 

XLVIII.  DISSOCIATION  PHENOMENA  IN  THE 

TOXIN-ANTITOXIN  COMBINATION Otto  and  Sachs.  666 

XLIX.  THE  PARTIAL  FUNCTIONS  OF  CELLS Ehrlwh.  676 

INDEX.  THE  H^MOLYTIC  AND  BACTERIOLYTIC  REACTIONS  DESCRIBED 

IN  THE   TEXT 695 

AUTHORITIES  QUOTED 697 

SUBJECTS . .  .  701 


COLLECTED    STUDIES  IN  IMMUNITY. 


I.  CONTRIBUTIONS  TO  THE    THEORY  OF  LYSIN 

ACTION.1 

By  Prof.  Dr.  P.  EHRLICH  and  Dr.  J.  MORGENROTH. 

ONE  of  the  most  important  advances  in  the  study  of  immunity 
is  the  discovery  of  Pfeiffer's  phenomenon,  and  it  is  to  Pfeiffer's 
splendid  observations  that  we  owe  the  first  and  most  important 
insight  into  the  mode  of  action  of  the  bacteriolytic  immune  sera. 

As  is  well  known,  the  phenomenon  of  bacteriolysis,  first  demon- 
strated by  Pfeiffer  in  a  guinea-pig  immunized  against  cholera,  con- 
sists in  the  immediate  dissolution  of  cholera  bacilli  introduced  into 
the  abdominal  cavity  of  the  animal.  The  same  takes  place  when 
the  bacilli  together  with  a  small  amount  of  immune  serum  are  intro- 
duced into  the  abdominal  cavity  of  a  normal  guinea-pig.  Subse- 
quently Metchnikoff  (Annal.  Inst.  Pasteur,  June  1895)  showed  that 
the  phenomenon  of  bacteriolysis  takes  place  also  outside  the  animal 
body,  in  vitro,  provided  a  small  quantity  of  peritoneal  exudate  of 
a  normal  guinea-pig  is  added.  Bordet  (Annal.  Inst.  Pasteur,  June 
1895)  was  thereupon  able  to  show  that  the  immune  serum  is  able 
to  effect  bacteriolysis  in  vitro  without  any  addition,  provided  that  it 
is  absolutely  fresh.  On  standing  it  becomes  inactive;  but  it  may 
be  reactivated  by  even  very  small  amounts  of  normal  serum.  Pfeiffer's 
ideas  as  to  the  nature  of  bacteriolysis  were  formulated  by  him  in  a 
very  clever  theory  which  he  published  in  1896  (Deutsche  med.  Wo- 

1  Reprinted  from  Berl.  klin.  Wochenschr.,  1899,  Xo.  1. 


2  COLLECTED  STUDIES  IN  IMMUNITY. 

chenschr.,  1896,  Nos.  7  and  8)  and  which  is  here  reproduced  only  in 
its  main  features. 

The  immunizing  substances  contained  in  cholera  serum  possess 
but  feeble  power  to  retard  development.  They  are  nothing  but  an 
antecedent  form  of  substances  developed  in  the  peritoneum  of  the 
guinea-pig,  specifically  solvent  for  cholera  vibrios.  They  are  stored 
in  the  animal  body  in  an  inactive  but  stable  form,  somewhat  as 
glycogen  is  stored  in  cell  depots  as  an  antecedent  form  of  grape- 
sugar.  When  needed,  these  inactive  substances  of  the  serum  can  be 
converted  into  the  specific  active  form  through  the  active  interference 
of  the  body-cells.  This  conversion  can  also  be  effected  by  the  addi- 
tion of  a  suitable  serum.  In  this  added  serum  a  certain  "something/' 
present  in  very  small  amounts,  effects  the  change,  but  is  very  soon 
used  up  in  the  process.  In  the  animal  body,  on  the  other  hand, 
this  constituent  is  produced  by  the  body-cells  as  long  as  the  stimulus, 
caused  by  the  presence  of  the  cholera  bacilli,  lasts.  The  action  of 
this  substance  is  ferment-like.  Bacteriolysis  is  also  regarded  as  a 
ferment  action,  caused  by  ferments  of  a  very  peculiar  kind.  These 
ferments  are  fitted  in  an  absolutely  specific  manner  each  to  a  single 
bacterial  protoplasm,  acting  on  this  exactly  as  pepsin  or  trypsin  acts 
on  coagulated  albumin.  According  to  Pfeiffer,  a  somewhat  distant 
analogy  is  seen  in  E.  Fischer's  yeast  ferments,  each  of  which  can  only 
split  up  a  sugar  of  a  definite  composition.  If  this  theory  be  correct, 
these  specific  ferments  must  exist  in  an  active  and  an  inactive  modi- 
fication. 

Recently  Bordet  (Annal.  Inst.  Pasteur,  Vol.  12,  No.  10)  pub- 
lished a  series  of  experiments  in  which  he  showed  that  the  laws  which 
govern  the  specific  bacteriolytic  action  of  immune  sera  govern  also 
certain  specific  solvent  phenomena  seen  in  red  blood-cells. 

Bordet  treated  guinea-pigs  with  repeated  injections  of  defibri- 
nated  rabbit  blood.  The  serum  of  animals  so  treated  possesses  the 
property  of  dissolving  rabbit  blood  in  vitro  rapidly  and  with  great 
intensity,  whereas  serum  of  normal  guinea-pigs  is  unable  to  do  this. 
Solution  is  preceded  by  a  marked  agglutination  of  the  erythrocytes. 
On  heating  the  specific  serum  for  half  an  hour  to  55°  C.  the  hsemolytic 
power  is  destroyed,  while  the  agglutinating  power  remains.  The 
serum  thus  inactivated  can  again  be  rendered  active  by  the  addition 
of  a  certain  amount  of  normal  guinea-pig  serum,  and  even  of  normal 
rabbit  serum.  The  active  guinea-pig  serum  has  no  effect  on  the 
red  blood- cells  of  the  guinea-pig  itself  or  on  those  of  pigeons,  but 


CONTRIBUTIONS  TO  THE  THEORY  OF  LYSIN  ACTION.          3 

acts,  though  to  a  less  degree,  on  the  blood-cells  of  rats  and  mice. 
The  active  guinea-pig  serum  injected  into  the  ear- vein  of  a  rabbit 
is  highly  toxic  to  that  animal. 

The  analogy  existing  between  these  phenomena  and  those  of 
bacteriolysis  is,  as  emphasized  by  Bordet,  a  very  close  one.  This 
will  be  clear  to  the  reader.  Very  likely,  therefore,  the  mechanism 
of  haemolysis  and  that  of  bacteriolysis  are  very  similar.  The  study 
of  haemolysis  thus  gains  considerable  theoretical  significance. 

Being  so  fortunate  as  to  have  at  our  disposal  a  considerable  amount 
of  appropriate  serum,  we  have  used  this  in  order  to  gain  a  deeper  in- 
sight into  the  nature  of  haemolysis.  This  serum  was  derived  from  a 
goat  which  during  eight  months  had  been  subcutaneously  injected  in 
somewhat  irregular  fashion  with  sheep  serum  rich  in  blood-corpuscles. 
The  experiments  were  therefore  made  with  sheep  blood  in  the  form 
of  a  5%  mixture  of  the  defibrinated  blood  in  0.85%  salt  solution. 
By  means  of  this  great  dilution  certain  sources  of  error  arising  from 
the  constituents  of  the  serum  are  avoided.  These  had  manifested 
themselves  in  Bordet's  experiments. 

The  serum  of  our  goat  rapidly  dissolves  sheep  blood-cells  in  vitro. 
The  degree  of  action  of  this  serum  can  be  accurately  determined  as 
follows:  To  each  5  cc.  of  the  above-mentioned  blood  mixture  decreas- 
ing amounts  of  the  goat  serum  are  added.  It  is  then  found  that 
at  37°  C.  the  specimens  containing  from  1.5  cc.  to  0.8  cc.  serum  will 
become  completely  laky.  After  allowing  all  the  specimens  to  act 
for  two  hours  in  a  thermostat  they  are  placed  in  a  refrigerator  and 
allowed  to  settle.  It  will  then  be  found  that  there  is  a  regular 
decrease  in  the  amount  of  solution  effected  until  finally  the  limit 
is  reached  in  the  specimen  containing  0.1  cc.  of  serum.  The  serum 
of  normal  goats  (we  tried  the  sera  of  a  number  of  different  animals) 
is  unable  even  in  large  amounts  to  dissolve  sheep  blood-cells.  It 
is  to  be  remarked  that  in  the  use  of  this  immune  serum  in  the  amounts 
mentioned  no  clumping  was  ever  observed  to  precede  haemolysis, 
although  this  phenomenon  was  carefully  looked  for.1 

1  The  serum  of  normal  goats  in  doses  of  1.5  cc.  and  over  possesses  the  prop- 
erty to  agglutinate  sheep  blood-cells,  but  this  property  seems  to  be  subject 
to  great  individual  and  chronologic  fluctuations.  This  agglutination  of  foreign 
bloods  by  certain  normal  sera,  and  which  probably  corresponds  to  the  normal 
agglutinating  action  of  sera  on  bacteria,  was  observed  many  years  ago  by 
Creite  (Z.  f.  rat.  Med.,  Vol.  36)  and  later  was  again  emphasized  by  Landois 
(Die  Transfusion  des  Blutes,  1875). 


4  COLLECTED  STUDIES  IX  IMMUNITY. 

If  the  immune  serum  is  heated  to  56°  C.,  it  completely  loses  its 
solvent  action.  The  addition  of  serum  of  normal  animals  to  this 
inactivated  serum  causes  it  to  be  reactivated.  For  this  purpose  one 
can  use  not  only  normal  goat  serum  but  also  normal  sheep  serum, 
though  the  latter  acts  somewhat  more  feebly.  This  power  of  the 
normal  serum  to  reactivate  an  inactive  immune  serum  is  very  readily 
lost.  Even  when  the  serum  is  kept  on  ice  and  protected  against 
light  it  very  soon  shows  a  diminution  of  its  reactivating  power.  In 
uantitative  experiments,  therefore,  the  inactive  (stable)  immune 
serum  should  always  be  reactivated  by  a  perfectly  fresh  normal 
serum. 

In  hsemolysis,  as  in  Pfeiffer's  bacteriolysis,  we  are  therefore  forced 
to  assume  the  existence  of  two  substances.  One  of  these,  specific 
and  quite  resistant  (stable),  we  shall  call  the  immune  body,  following 
Pfeiffer's  nomenclature.  The  other,  normally  present  and  highly 
labile  (unstable),  we  shall  for  the  present  term  addiment. 

Although  our  results  in  the  main  agree  with  those  of  Bordet, 
we  must  at  once  call  attention  to  one  difference  in  our  observations. 
As  already  mentioned,  the  action  of  our  goat  serum  on  the  sheep 
blood-cells  is  not  preceded  by  any  agglutination.  From  this  we  see 
that  the  agglutination  cannot  be  considered  a  preparatory  step  neces- 
sary for  the  hsemolytic  action,  as  Bordet  seems  to  assume.  The 
specific  agglutinin  has  no  relation  whatever  to  the  hsemolytic 
immune  body.  Similarly,  according  to  the  views  of  eminent  bacte- 
riologists, the  specific  bacteriolytic  substances  have  no  relation  to 
the  agglutinins.  The  lysins  may  exist  independently  of  the  agglu- 
tinins  and  these  again  independently  of  the  bacterioloytic  substances. 
The  reader  is  reminded  of  the  interesting  observations  of  Pfeiffer 
and  Kolle.  These  investigators  described  an  immune  serum  which 
was  strongly  bacteriolytic  but  which  did  not  at  all  agglutinate  (Cen- 
tralblatt  f.  Bakt.,  1896,  Vol.  XX,  Nos.  4  and  5).  On  the  other  hand, 
E.  Frankel  and  Otto  state  that  if  a  young  dog  be  fed  on  typhoid 
cultures,  the  dog's  serum  will  acquire  agglutinating  but  not  bacte- 
riolytic properties.  Similarly,  if  a  frog  is  treated  with  typhoid  bacilli, 
the  frog  serum  will  agglutinate  such  bacilli.  They  remain  in  the 
lymph  sac  of  the  animal,  however,  not  only  alive  but  virulent.  (Widal 
and  Sicard,  Comptes  rend.  Soc.  de  BioL,  XI.  27-97). 

Pfeiffer's  original  theory  sought  only  to  explain  in  general  the 
mode  of  action  of  the  specific  bacteriolysins.  It  did  not  concern 
itself  with  the  questions  how  or  where  they  originated.  It  was  in 


CONTRIBUTIONS  TO  THE  THEORY  OF  LYSIN  ACTION.          5 

order  to  throw  some  light  on  these  problems  that  Ehrlich  devised 
his  side-chain  theory. 

At  first  Ehrlich's  theory  was  applied  to  the  origin  of  the  anti- 
toxins and  to  the  chemical  relation  existing  between  the  toxins  and 
certain  atomic  groups  of  the  protoplasmic  molecule.  Pfeiffer  him- 
self applied  the  theory  to  the  substances  specifically  bacteriolytic 
for  cholera  bacilli,  and  was  able  to  demonstrate  experimentally  that 
the  source  of  the.-e  bodies  was  in  the  spleen,  the  bone-marrow,  and  the 
lymph  bodies  (Pfeiffer  and  Marx,  Zeitschr.  f.  Hyg.,  Vol.  37,  1898). 
Wu.ssermann,  who  in  his  well-known  tetanus  experiments  had  fur- 
nished the  first  demonstration  of  the  soundness  of  the  side-chain 
theory,  succeeded  in  showing  the  source  of  the  specific  typhoid 
bacteik)lysin.  The  study  of  these  bacteriolytic  processes  brought  up 
a  number  of  important  questions  directly  concerning  the  side-chain 
theory,  and  we  felt  compelled  to  examine  these  experimentally. 

According  to  Ehrlich's  theory,  if  any  substance,  be  it  toxin,, 
toxoid,  ferment,  or  constituent  of  a  bacterial  cell  or  of  a  blood- 
corpuscle,  possess  the  property  of  combining  with  side-chains  of  the 
protoplasm,  the  possibility  is  given  for  the  formation  of  a  corre- 
sponding antibody.  The  antibody,  according  to  the  theory,  must 
possess  such  a  group  as  will  fit  the  haptophore  (the  specific  com- 
bining) group  of  the  invading  substance.  The  soluble  body,  therefore, 
produced  in  response  to  the  invading  substance  (toxin,  toxoid,  etc), 
must  combine  chemically  with  the  latter.  If  the  invading  substance 
is  in  soluble  form,  as,  for  example,  the  toxins,  the  neutralization 
proceeds  in  the  solution.  If,  however,  it  is  not  directly  soluble, 
being  originally  an  insoluble  part  of,  say,  a  bacterial  or  blood  cell, 
then  the  dissolved  antibody  in  the  blood  will  be  abstracted  from 
its  solvent  fluid  and  anchored  by  the  cell  particle.  In  the  well-known 
experiment  of  Wassermann  on  tetanus  poison,  the  same  thing  is  seen. 
In  this  the  invading  substance  (tetanus  toxin)  is  abstracted  from 
its  solution  and  anchored  by  the  crushed  brain  cells.  In  order  to 
maintain  the  analogy  we  should  expect  that  in  our  experiment  the 
immune  body  dissolved  in  the  goat  serum  would  be  anchored  by  the 
erythrocytes  of  sheep  blood. 

The  manner  of  procedure  in  this  experiment  is  very  simple  and 
consists  in  the  addition  to  sheep  blood,  or  a  dilution  of  the  same, 
of  immune  serum  which  has  been  heated  to  56°  C.  in  order  to  destroy 
its  solvent  properties.  The  mixture  is  then  centrifuged  to  separate 
the  cells  and  the  fluid.  In  case  the  immune  body  has  been  anchored 


6  COLLECTED  STUDIES  IN   IMMUNITY. 

by  the  blood-cells,  the  clear  fluid  should  be  free  from  the  same.  To 
prove  this  we  have  merely  to  add  to  some  of  this  clear  fluid  sheep 
blood-cells,  and  a  sufficient  amount  of  addiment  in  the  form  of  normal 
serum.  If  the  fluid  is  free  from  immune  body,  the  blood-cells  will 
remain  undissolved.  The  centrifuged  sediment  must  likewise  be 
tested  for  the  presence  of  immune  body.  The  sediment,  freed  as 
much  as  possible  from  fluid,  is  mixed  with  salt  solution  and  a  suffi- 
cient amount  of  addiment.  If  a  corresponding  amount  of  immune 
body  has  been  anchored  by  the  blood-cells,  they  will  now  dissolve. 
One  of  our  numerous  experiments  follows: 

4  cc.  of  a  5%  mixture  of  sheep  blood-cells  are  mixed  with  1.0 
or  1.3  cc.  inactivated  serum  from  our  immunized  goat.  This  is  allowed 
to  stand  for  fifteen  minutes  at  40°  C.  and  then  carefully  centrifuged. 
The  supernatant  clear  fluid  is  poured  off,  mixed  with  0.2  cc.  normal 
sheeps  blood  and  then  with  0.8  cc.  serum  from  a  normal  goat.  This 
mixture  after  being  kept  in  a  thermostat  at  37°  C.  for  two  hours 
and  then  allowed  to  settle  in  the  cold,  shows  no  trace  of  solution. 

The  centrifuged  sediment,  freed  as  much  as  possible  from  fluid 
by  means  of  filter  paper,  is  mixed  with  4  cc.  physiological  salt  solu- 
tion and  with  0.8  cc.  normal  goat  serum.  This  mixture  after  being 
kept  for  two  hours  in  a  thermostat  at  37°  C.  is  found  completely 
dissolved  or  very  nearly  so. 

In  this  experiment  in  which  a  sufficient  amount  of  immune  body 
was  used,  we  see  that  complete  union  took  place  between  the  immune 
body  and  the  blood-cells,  resulting  in  the  entire  abstraction  of  the 
former  from  the  fluid.  We  have  found  that  the  same  takes  place 
at  lower  temperatures,  even  at  0°  C.  That  this  is  a  chemical  union 
and  not  a  mere  absorption  is  seen  by  experiments  with  other  species 
of  blood.  Thus  the  red  blood-cells  of  rabbits  and  of  goats  have  no 
affinity  whatever  for  this  immune  body. 

As  a  result  of  these  experiments,  therefore,  and  in  conformity  with 
the  side-chain  theory,  we  must  assume  that  the  immune  body  possesses 
a  specific  haptophore  group  which  anchors  it  to  the  blood-cells  of  the 
sheep. 

The  next  important  question  was  that  concerning  the  relation 
of  the  addiment  to  the  red  blood-cell.  This  was  studied  in  a  manner 
exactly  similar  to  that  of  the  previous  experiment.  Blood  was 
mixed  with  addiment,  the  mixture  centrifuged,  and  the  two  por- 
tions tested  separately,  by  the  addition  of  immune  body,  for  the 
presence  of  addiment.  We  varied  our  experiments  greatly  so  far 


CONTRIBUTIONS  TO  THE  THEORY  OF  LYSIN  ACTION.          7 

as  time  and  temperature  conditions  were  concerned,  but  the  result 
was  always  the  same;  the  red  blood-cells  did  not  combine  with  a  trace 
of  addiment.  This  is  in  direct  contrast  to  their  behavior  toward  the 
immune  body. 

Having  now  determined  the  behavior  of  the  blood-cells  to  immune 
body  and  addiment  separately,  it  remained  to  see  what  the  affinities 
of  the  blood-cells  were  when  both  of  these  bodies  were  present  at  the 
same  time.  The  solution  of  this  problem  offers  many  technical 
difficulties.  Practically  it  will  be  best  to  make  the  mixtures  so  that 
there  will  be  just  the  proper  amount  of  the  two  ingredients  to  effect 
complete  solution  of  the  blood-cells.  We  found  that  if  we  mixed 
1.0  to  1.3  cc.  of  our  inactivated  goat  serum  with  0.5  cc.  normal  goat 
serum,  this  would  just  suffice  to  dissolve  5  cc.  of  a  5^  mixture  (in 
saline)  of  sheep  blood-cells.  If  this  mixture  is  placed  in  the  ther- 
mostat, complete  solution  will  ensue;  but  because  an  excess  of  the 
solvent  substances  has  been  avoided,  the  process  does  not  take  place 
rapidly.  Usually  it  is  completed  at  the  end  of  H  to  2  hours. 

If  the  mixture  is  kept  at  0°-3°  C.,  no  solution  occurs,  and  if  it  is 
then  centrifuged  and  examined  according  to  the  methods  just  studied, 
the  red  blood-cells  will  be  found  to  have  loaded  themselves  with 
immune  body,  leaving  the  addiment  in  the  fluid.  The  experiment 
shows  that  under  the  conditions  mentioned,  addiment  and  immune 
body  exist  in  the  fluid  entirely  independent  of  one  another. 

It  still  remained  to  determine  the  combining  affinities  at  higher 
temperatures.  A  preliminary  trial  showed  that  if  we  used  the  pro- 
portions above  mentioned  and  kept  such  mixtures  hi  an  Ostwald 
water-bath  at  40°  C.  for  six,  ten,  thirteen,  and  eighteen  minutes 
respectively  and  then  centrifuged,  only  in  the  first  two  tubes  did 
the  fluid  remain  colorless,  while  hi  the  other  tubes  it  was  distinctly 
red.  In  the  experiments  at  this  temperature  we  therefore  adopted 
a  time  limit  of  ten  minutes.  A  tube  of  the  above-mentioned  mixture 
was  allowed  to  remain  in  the  water-bath  at  40°  C.  for  ten  minutes 
and  then  centrifuged.  The  results  were  as  follows: 

The  sediment  mixed  with  salt  solution  shows  haemolysis  of  a 
moderate  degree.  (This  occurs  even  if  the  sediment  is  mixed  with 
ice-cold  salt  solution,  centrifuged,  and  then  again  mixed  with  salt 
solution.  By  this  manipulation  the  last  trace  of  fluid  originally 
adhering  to  the  cells  is  removed.)  Solution  becomes  complete  when 
new  addiment  in  the  form  of  normal  serum  is  added  to  the 
mixture.  The  centrifuged  fluid  does  not,  by  itself,  dissolve  blood 


8  COLLECTED  STUDIES  IN  IMMUNITY. 

added  to  it,  or  it  does  so  in  only  a  very  limited  degree.  When,  how- 
ever, new  immune  body  is  added,  the  blood-cells  are  completely  dis- 
solved. 

From  these  experiments  we  conclude  that  the  sediment  this  time 
contained  both  components,  though  not  in  equivalent  proportion, 
for  there  was  an  excess  of  immune  body  which  became  manifest 
only  on  the  addition  of  new  addiment.  Corresponding  to  this  the 
centrifuged  fluid  contained  only  faint  traces  of  immune  body  and  an 
excess  of  addiment. 

The  explanation  of  these  phenomena  presents  no  difficulties.  It 
must  be  assumed  that  under  certain  circumstances  the  immune  body 
and  addiment  enter  into  loose,  readily  dissociated  chemical  combi- 
nation. This  combination  is  hastened  by  heat  and  retarded  by  cold 
in  entire  conformity  to  the  views  previously  expressed  by  Ehrlich 
(Werthbemessung  des  Diphtherie-heilserums,  Jena,  1897).  On  the 
other  hand,  the  affinity  existing  between  blood-cells  and  immune 
body  must  be  very  strong,  for  these  combine  completely  even  in  the 
cold.  We  must  therefore  assume  that  the  immune  body  possesses  two 
different  haptophore  groups,  one  with  a  strong  affinity  for  the  corre- 
sponding haptophore  group  of  the  red  blood-cell,  and  the  other  of  feeble 
chemical  affinity,  which  is  able  to  combine  more  or  less  completely  with 
the  addiment  present  in  the  serum.  At  30°  C.,  therefore,  the  red  blood- 
cell  attracts  to  itself  not  only  the  free  molecules  of  immune  body, 
but  also  those  which  have  already  combined  with  the  addiment  in 
the  fluid.  In  the  latter  case  the  immune  body  represents  in  a  measure 
a  link  which  ties  addiment  to  the  red  blood-cells  and  subjects  these 
to  the  action  of  the  addiment.  In  agreement  with  Pfeiffer,  we  regard 
the  phenomena  appearing  under  the  influence  of  the  addiment  as 
analogous  to  digestion,  and  we  shall  probably  not  err  if  we  regard  the 
addiment  as  having  the  character  of  a  digestive  ferment.  Morgen- 
roth,  by  the  experiments  in  which  by  immunization  he  successfully 
produced  an  antibody  against  rennin  ferment,  has  made  it  very 
probable  that  the  ferments,  like  the  toxins,  possess  two  groups, 
one  a  haptophore  group  and  the  other  the  actual  carrier  of  the  fer- 
ment action. 

With  this  preliminary  analysis  all  the  various  phenomena  are 
now  readily  explained.  We  assume  that  the  immune  body  combines 
with  the  small  amount  of  digesting  ferment  normally  present  in 
the  blood,  and  then,  by  means  of  its  other  haptophore  group,  fitting, 
for  example,  to  red  blood-cells  or  bacteria,  carries  this  digestive 


CONTRIBUTIONS  TO  THE  THEORY  OF  LYSIN  ACTION. 

action  over  to  these  cells.  From  this  we  see  also  why  the  digestive 
action  becomes  manifest  only  on  the  addition  of  immune  body. 
This  brings  the  ferment,  present  in  the  serum  fluid  in  such  small 
quantity,  to  the  blood-cells  in  comparatively  large  amounts,  thur:- 
concentrating  and  increasing  its  action.  It  is  possible  and  even 
probable  that  only  a  few  substances  with  digestive  properties  exist 
in  the  blood,  perhaps  only  one;  but  that  a  countless  variety  of  specific 
immune  bodies  can  exist  there,  as  Gruber,  among  others,  assumes. 
In  that  case  we  must  assume  that  in  these  immune  bodies  there  is 
always  one  group  which  fits  only  to  the  cells  or  substances  used  to 
excite  its  production,  but  that  all  these  immune  bodies  possess  an 
atomic  group  in  common  which  effects  the  combination  with  the 
digestive  substance.  On  this  assumption  it  is  very  easy  to  explain 
by  means  of  the  side-chain  theory  the  otherwise  difficult  problem 
of  the  mode  of  origin  of  the  lysins.  According  to  Ehrlich's  definition, 
the  side-chains  possess  definite  atomic  groups  which  are  able  to  com- 
bine with  certain  other  atomic  groups  and  so  increase  the  proto- 
plasmic molecule.  As  far  back  as  1885  (Sauerstoff  Bediirfniss  des 
Organismus)  Ehrlich  had  pointed  out  that  the  atomic  groups  thus 
anchored  to  the  living  substance  were  much  more  readily  oxidized 
and  that  they  therefore  represent  the  nourishment  (KCXT  egoxrfv}  of 
the  cell.  The  study  of  immunity  has  considerably  extended  this  view 
and  taught  us  that  the  antibody  represents  such  thrust-off  side- 
chains;  further,  that  the  immunizing  process  consists  in  forcing  the 
particular  organism  to  produce  these  side-chains  in  surplus  amount 
in  conformity  with  Weigert's  theory  of  cell  injury.  It  is  of  course 
very  probable  that  these  side-chains,  according  to  their  special  func- 
tion, will  be  differently  constituted.  If  a  side-chain  is  designed 
to  assimilate  relatively  simple  substances,  we  may  believe  that  the 
possession  of  a  single  combining  group  will  suffice.  Very  likely  the 
side-chains  which  anchor  toxins  are  of  this  simple  type.  Eut  it  is 
entirely  different  wrhen  a  giant  molecule  (albumin  molecule)  is  to  be 
assimilated.  In  this  case  the  anchoring  of  the  molecule  is  only  a  pre- 
liminary requisite.  Such  a  giant  molecule  is  useless  to  the  cell  and 
can  only  then  be  utilized  when  it  is  broken  up  by  fermentative  pro- 
cesses into  smaller  parts.  It  will  be  particularly  advantageous  to 
the  cell  if  its  "grasping  arm"  is  at  the  same  time  a  carrier  of  a  fer- 
mentative group  which  can  at  once  be  brought  to  bear  on  the  anchored 
molecule.  We  see  such  well-adapted  contrivances  (in  widen  the 
grasping  apparatus  also  possesses  digesting  properties)  in  a  whole 


10  COLLECTED  STUDIES  IN  IMMUNITY. 

-series  of  higher  plants.  For  example,  the  tentacles  of  Drosera,  which 
may  be  regarded  as  grasping  arms  in  the  widest  sense,  secrete  a  strong 
digesting  fluid. 

If,  then,  we  see  that  lysin  action  does  not  occur  with  toxins,  but 
only  when  the  contents  of  cells  are  absorbed,  be  these  bacteria  or 
blood-cells,  we  must  conclude  that  in  the  latter  case  large-moleculed 
albuminous  substances  are  concerned.  These  are  much  more  complex 
in  structure  than  the  toxins,  which  represent  mere  cell  secretions. 
For  the  assimilation  of  the  highly  complex  bodies  we  therefore  assume 
the  existence  of  side-chains  of  a  peculiar  kind.  These,  besides  their 
combining  group,  possess  another  group  which  by  fixation  with 
special  ferments  causes  the  digestion  of  the  complex  substances.  If, 
by  means  of  the  immunizing  process,  one  succeeds  in  having  a  surplus 
of  these  side-chains  produced,  they  will  be  produced  with  both  these 
functional  groups  and  thrust  off  into  the  blood  as  immune  body. 
This  explains  the  wonderful  contrivance  whereby  the  injection  of  a 
bacterium  is  followed  by  the  production  of  a  substance  which  destroys 
this  bacterium  by  dissolving  it.  This  phenomenon  is  nothing  but 
the  reproduction  of  a  process  of  normal  cell  life. 


n.  CONCERNING   ILEMOLYSINS.1 
SECOXD  COMMUNICATION. 

By  Professor  Dr.  P.  EHRLICH  and  Dr.  J.  MORGENROTH. 

IN  a  previous  paper2  we  demonstrated  the  relations  existing 
between  the  red  blood-cells  to  be  dissolved  and  the  two  components  of 
a  specific  haemolysin  produced  by  immunization.  It  will  be  remem- 
bered that  we  termed  the  two  components  of  the  specific  serum 
immune  body  and  addiment.  We  were  able  to  show  that  the  immune 
body  combines  with  the  erythrocytes  of  the  species  whose  blood 
was  injected,  since  it  has  a  specific  affinity  for  these  cells.  We  showed 
further  that  the  addiment,  the  unstable  (labile)  ferment-like  body 
which  effects  the  solution  of  the  blood-cells,  is  tied  to  these  cells 
indirectly  by  means  of  the  immune  body. 

Proof  was  thus  afforded  that,  in  conformity  with  the  require- 
ments of  the  side-chain  theory,  the  immune  body  possesses  one 
haptophore  group  by  means  of  which  it  combines  with  the  erythrocytes 
of  the  corresponding  blood,  and  a  second  haptophore  group  with  less 
affinity  by  which  it  combines  with  the  addiment  and  transfers  the 
action  of  the  latter  to  the  blood-cells. 

At  that  time  we  availed  ourselves  of  the  serum  of  a  goat  which 
had  been  treated  for  some  time  with  subcutaneous  injections  of  a 
sheep  serum  rich  in  blood  corpuscles.  Corresponding  to  this  treat- 
ment, the  serum  of  the  goat  possessed  a  moderate  degree  of  solvent 
action  on  sheep  blood-cells. 

In  order  to  continue  these  studies  it  seemed  essential  to  make 
use  of  a  serum  derived  from  an  animal  treated  for  some  time  with 
full  blood,  a  serum  that  would  accordingly  possess  a  higher  degree 
of  activity.  For  this  purpose  we  began  the  immunization  (Nov.  12 

1  Reprinted  from  Berl.  klin.  Wochenschr.  1899,  No.  22. 

2  See  pages  1-10  of  this  volume. 

11 


12  COLLECTED  STUDIES  IN  IMMUNITY. 

and  Feb.  24)  of  two  male  goats  by  injecting  them  subcutaneously 
with  increasing  amounts  of  defibrinated  sheep  blood.  In  a  short 
time  a  strongly  active  serum  was  produced  in  both  animals,  and 
we  were  able  to  observe  how,  following  the  general  laws  of  immu- 
nization, its  activity  increased.  The  course  of  the  immunization 
did  not  manifest  any  peculiarities.  It  should,  however,  be  remarked 
that  on  the  days  following  the  injection  of  a  considerable  amount 
of  blood  (350  cc.)  not  the  least  decrease  in  the  activity  of  the  serum 
could  be  observed,  in  contrast  to  the  experiences  with  tetanus  or 
diphtheria  immunization. 

So  far  as  the  general  method  employed  in  the  following  experi- 
ments is  concerned,  it  was  the  same  as  that  mentioned  in  the  first 
paper.  The  blood  was  always  used  in  the  form  of  a  5%  suspension 
in  physiological  salt  solution.  At  the  time  of  these  experiments 
the  serum  of  buck  I  was  able  to  dissolve  the  sheep  blood  com- 
pletely in  the  proportion  of  0.2-0.3  cc.  serum  to  5  cc.  sheep  blood 
mixture;  0.03-0.07  cc.  serum  were  able  to  produce  a  just  noticeable 
amount  of  solution.  Of  the  serum  of  buck  II,  0.15-0.2  cc.  suf- 
ficed for  complete  solution.  It  should  be  mentioned  that  the  serum 
of  buck  II  even  before  immunization  possessed  a  slight  solvent 
effect  on  sheep  blood.  This,  however,  was  so  slight  that  4.0  cc.  of 
the  serum  were  not  nearly  able  to  dissolve  5  cc.  of  the  5%  blood 
mixture,  and  1.2  cc.  serum  produced  only  a  just  noticeable  amount 
of  solution.  Heating  the  serum  to  57°  C.  for  half  an  hour  destroyed 
this  action,  as  it  did  also  that  for  rabbit  and  guinea-pig  blood.1 

With  the  sera  of  these  two  bucks  we  were  now  able  to  proceed 
with  our  experiments.  The  combination  of  the  immune  body  with 
the  erythrocytes  of  the  sheep  at  0°  C.  can  be  readily  demonstrated, 
for  at  this  temperature  and  by  the  employment  of  proper  amounts 
of  serum  no  solution  takes  place.  The  serum  was  allowed  to  act  on 
the  sheep  blood  for  twenty-four  hours,  care  being  taken  to  keep  the 
mixture  at  0°  C.  The  blood-cells  were  then  separated  by  means 


1  On  examining  the  sera  of  a  large  number  of  normal  goats  one  will  find 
some  sera  which  possess  this  feeble  solvent  power  for  sheep  blood.  Thus  the 
normal  goat  sera  which  we  employed  for  control  tests  in  our  first  experiments, 
and  which  were  used  in  great  number,  failed  absolutely  to  show  any  solvent 
action,  but  at  most  manifested  only  a  variable  degree  of  agglutinating  action. 
This  will  be  seen  from  our  reports  at  that  time.  In  our  first  communication 
we  had  already  called  attention  to  the  great  variability  of  the  agglutinating 
property. 


CONCERNING  ILUMOLYSINS.  13 

of  the  centrifuge,  and  they  showed  by  their  behavior  that  they  had 
combined  with  the  immune  body.  They  did  not  dissolve  on  the 
addition  of  physiological  salt  solution,  but  dissolved  when  addiment 
in  the  form  of  normal  goat  serum  was  added.  In  contrast  to  this, 
both  components  combined  with  the  sheep  blood-cells  when  the 
mixture  was  kept  at  room  temperature  (about  20°  C.)  even  for  only 
eight  minutes.  The  blood-cells,  separated  by  centrifuge  and  washed 
with  physiological  salt  solution  to  free  them  from  traces  of  serum, 
were  mixed  with  more  salt  solution  and  placed  in  an  incubator, 
where  they  dissolved  in  considerable  quantity. 

These  new  and  stronger  immune  sera  therefore  exhibited  proper- 
ties in  relation  to  the  sheep  blood-cells  entirely  analogous  to  those 
of  the  serum  previously  described  by  us.  On  the  other  hand  in  cer- 
tain respects  their  behavior  was  entirely  different. 

The  serum  described  by  Bordet,  as  well  as  that  of  our  goats,1  lost 
its  haBinolytic  power  when  heated  for  half  an  hour  to  56°  C.  This  has 
been  shown  by  Buchner  to  be  true  of  all  normal  hsemolytic  sera. 
The  sera  of  our  two  bucks  even  when  heated  for  three-quarters  of  an 
hour  to  56°  C.  showed  only  a  scarcely  appreciable  diminution  of  their 
solvent  action  on  sheep  blood,  while  their  normal  solvent  action  on  guinea- 
pig  blood  and  rabbit  blood  was  entirely  destroyed.  Even  when  the  serum 
was  heated  to  56°  C.  for  three  hours  or  when,  after  mixing  with  equal 
parts  of  water,  it  was  heated  for  one  and  one-half  hours  to  65°  C., 
it  showed  merely  a  reduction  in  its  solvent  action  for  sheep  blood, 
but  not  a  destruction  of  this  action. 

Our  preliminary  experiments  on  the  combining  relations  had 
shown  us  that  the  action  of  these  hsemolysins  was  due  to  the  pres- 
ence in  the  serum  of  a  specific  immune  body  and  an  addiment.  It  was 
therefore  clear  that  we  were  here  dealing  with  an  addiment  of  a  very 
peculiar  kind,  which  was  distinguished  from  the  addiments  of 
all  hsemolysins  heretofore  known  by  its  extraordinary  resistance 
to  thermic  influences.  This  property  must  pertain  to  the  addi- 
ment itself  and  cannot  be  ascribed  to  the  presence  of  another  sub- 
stance in  the  serum  increasing  its  resistance,  for  such  a  substance 
would  have  served  to  protect  the  haemolytic  bodies  normally  present. 

In  order,  however,  to  analyze  these  phenomena  completely,  it 
was  absolutely  essential  to  obtain  the  two  components  of  the  complex 


1  This  refers  to   the  female  goats.     The  male  goat   is  always  designated 
"buck"  by  Ehrlich  and  Morgenroth.     [Translator.] 


14  COLLECTED   STUDIES  IN   IMMUNITY. 

serum,  the  immune  body  as  well  as  the  addiment,  in  a  free  state. 
In  the  ordinary  specific  haemolytic  serum  the  former  is  usually  readily 
obtained  because  the  addiment  is  destroyed  by  slight  heating.  In 
the  case  of  our  serum,  however,  heating  proved  ineffective,  so  it 
became  necessary  to  adopt  other  means.  Experience  having  taught 
us  that  the  addiment  is,  as  a  rule,  more  readily  destroyed  than  the 
immune  body,  we  could  expect  to  accomplish  our  purpose  by  using 
stronger  destructive  agents  of  a  chemical  nature.  After  a  number 
of  trials  we  have  finally  made  use  of  the  following  procedure: 
One  part  of  our  serum  is  mixed  with  one-tenth  part  normal  hydro- 
chloric acid,  the  mixture  digested  at  37°  C.  for  30  to  45  minutes, 
and  then  neutralized.  It  will  be  found  that  the  serum  has  then  lost 
its  solvent  power  for  sheep  blood-cells;  but  that  it  still  possesses 
immune  body  in  scarcely  decreased  amount  can  be  shown  by  re- 
activating the  serum. 

The  isolation  of  the  immune  body  made  it  possible  finally  to  demon- 
strate the  combination  of  the  immune  body  at  higher  temperatures,  20°- 
35°  C.  This  combination  is  seen  to  be  quantitative,  i.e.,  the  sheep  blood- 
cells  are  able  to  combine  with  all  the  immune  body  present  in  that  quan- 
tity of  serum  which  in  its  active  state  would  just  suffice  for  their  com- 
plete solution.  For  example,  to  5  cc.  of  the  5%  blood  mixture,  0.15  cc. 
of  the  serum  inactivated  with  hydrochloric  acid  is  added,  it  having 
been  previously  ascertained  that  this  amount  of  active  serum  just 
suffices  for  complete  solution.  The  mixture  is  allowed  to  stand 
for  half  an  hour  at  room  temperature  and  is  then  centrifuged.  To 
the  sediment  2.0  cc.  normal  goat  serum  are  added,  and  to  the  clear 
fluid  some  additional  sheep  blood  mixture  and  2.0  cc.  normal  goat 
serum.  The  sediment  thus  treated  will  be  seen  to  dissolve  com- 
pletely, whereas  the  blood-cells  added  to  the  clear  fluid  remain  intact 
despite  the  presence  of  the  addiment.  This  shows  that  all  the  im- 
mune body  combined  with  the  sedimented  sheep  blood-cells. 

The  addiment  necessary  for  this  reactivation  is  present  in  normal 
goat  serum,  as  can  be  seen  from  the  experiment.  This  is  true  for  all 
goat  sera  thus  far  examined  by  us,  although  the  amount  varies. 
It  will  be  recalled  that  we  had  found  the  original  addiment  which  fitted 
the  immune  body  was  able  to  withstand  heat.  The  question  there- 
fore at  once  arises  whether  normal  serum  also  contains  such  heat- 
resisting  addiments.  As  a  matter  of  fact  this  was  found  to  be  the 
case  in  a  number  of  goats  examined  by  us.  When  the  serum  of  these 
goats  was  heated  for  i  to  J  hr.  to  56°  C.  and  its  normal  hsemolytie 


CONCERNING  H.EMOLYSIXS.  15 

properties  for  other  blood-cells  were  entirely  destroyed,  it  was  still 
able  to  typically  reactivate  the  particular  immune  body  here  con- 
cerned.1 In  another  series  of  goats*  however,  the  result  was  different, 
for  heating  the  serum  to  56°  C.  destroyed  its  reactivating  properties 
completely.  These  sera  then  contained  exclusively  a  thermolabile 
addiment  which,  like  the  thermostabile  addiment,  fitted  the  immune 
body.  We  must  therefore  conclude  that  the  immune  body  developed 
by  this  immunization  is  capable  of  being  activated  by  addiments 
of  two  kinds,  which  differ  from  each  other  by  their  resistance 
to  thermic  influences  and  which  are  both  present  in  normal  serum. 

It  is  probable  that  both  kinds  of  addiment  can  be  present  in 
goat  serum  at  the  same  time,  but  that  in  most  cases  only  one,  the 
thermolabile,  is  present.  The  varying  behavior  toward  thermic  in- 
fluences, manifested  by  the  sera  of  our  immunized  animals,  would  thus 
be  easily  explained.  We  assume  that  the  same  immune  body  was 
present  in  both  cases,  but  that  the  serum  of  the  goat  first  immunized  con- 
tained only  the  thermolabile  addiment,  while  the  sera  of  the  animals 
examined  later  contained  also  the  thermostabile  addiment.  In  this 
connection,  the  fact  that,  previous  to  the  commencement  of  immu- 
nization, we  were  able  to  demonstrate  a  considerable  content  of 
thermostabile  addiment  in  the  serum  of  the  third  animal  (buck  II) 
is  of  considerable  interest. 

Having  thus  arrived  at  some  understanding  of  the  action  of  the 
hsemolytic  sera  produced  by  immunization  it  seemed  essential  that 
we  extend  our  investigations  to  the  hamolytic  properties  of  normal 
sera.  These  properties  had  long  been  known  and  had  been  studied 
particularly  by  Buchner  and  his  pupils.2 

The  fact  that  the  hsemolytic  action  of  normal  serum  is  destroyed 
by  moderate  heat  led  us  to  believe  that  the  normal  hsemolysins  are 


1  As  it  is  thus  possible  to  destroy  all  the  normal  lysins  (which  interfere  with 
the  experiment)  it  ought  to  be  possible  to  determine  whether  a  similar  heat- 
resisting  addiment  also  occurs  in  the  serum  of  other  species.     We  succeeded 
in  demonstrating  its  presence  in  varying  amounts  in  the  serum  of  a  sheep  and 
of  a  calf,  but  failed  to  find  it  in  serum  of  a  dog  or  rabbit. 

2  It  is  very  probable  that  certain  forms  of  hapmoglobinuria  originate  through 
analogous  haemolysins.     Many  years  ago  Ehrlich  showed  that  the  hremoglobi- 
nuria  ex  frigore  was  caused,  not  by  any  particular  sensitiveness  of  the  erythro- 
cytes  to  cold,  but  by  certain  poisons  produced,  especially  by  the  vessels,  as  a 
result  of  the  cold.     Possibly  also  such  autolysins  play  an  important  role  in. 
the  convalescence  of  severe  anaemias. 


16  COLLECTED  STUDIES  IN  IMMUNITY. 

not  of  simple  constitution;  but  the  experimental  solution  of  this 
problem  was  attended  with  great  difficulties. 

The  primary  tests  necessary  to  demonstrate  the  complex  con- 
stitution of  a  lysin  are  very  readily  made  on  a  number  of  series. 
They  consist  in  this,  that  a  serum  which  dissolves  certain  red  blood- 
cells  at  ordinary  temperatures  is  mixed  with  these  cells  at  0°  and 
allowed  to  act  at  this  temperature  for  some  time.  For  example, 
goat  serum  is  mixed  with  guinea-pig  blood-cells,  for  which  it  is  nor- 
mally hsemolytic.  The  mixture  is  kept  at  0°  and  then  centrifuged. 
The  clear  fluid  is  mixed  with  an  additional  amount  of  blood-cells 
and  tested  in  the  usual  manner  for  its  hsemolytic  power.  In  this 
way  it  was  easily  shown  that  through  this  procedure  the  serum  had 
lost  part  of  its  power,  but  that  this  was  completely  restored  by 
the  addition  of  some  of  the  same  serum  previously  inactivated  by 
heat.  According  to  our  previous  experience  these  experiments  show 
that  this  serum  contains  two  substances:  one,  which  we  shall  call 
interbody,  possessing  two  haptophore  groups  and  analogous  to  the 
immune  body;  the  other,  an  addiment,  which  we  shall  hereafter  term 
complement.  Further,  they  show  that  of  these  two  bodies  the  blood- 
cells  combine  preponderantly  with  the  interbody.  The  decrease  in 
the  power  of  the  serum  is  thus  explained  by  a  lack  of  interbody, 
and  this  is  supplied  by  the  addition  of  inactive  serum. 

In  experiments  of  this  kind  we  have  succeeded  with  the  following 
combinations :  goat  serum,  sheep  serum,  calf  serum,  and  dog  serum, 
with  guinea-pig  blood. 

Although  the  demonstration  of  the  lack  of  interbody  is 
extremely  simple,  the  counter-demonstration,  that  this  interbody 
has  combined  with  the  sedimented  blood-cells,  is  extraordinarily 
difficult;  for  in  this  demonstration  a  completely  isolated  comple- 
ment is  essential.  The  production  of  a  complement  to  fit  the  specific 
interbody  obtained  by  heating  the  serum  of  our  immunized  goat 
is  extremely  easy,  for  it  is  found  in  all  normal  goat  serum  and  can 
also  be  obtained  from  immune  serum  by  means  of  elective  absorp- 
tion. 

It  will  be  well  to  analyze  the  conditions  governing  this  elective 
absorption  by  means  of  which  interbody  and  complement  can  be 
separated.  Complete  separation  will  be  possible  when,  under  the 
circumstances  prevailing  at  the  time,  the  affinity  of  the  interbody 's 
haptophore  group  for  the  blood-cells  is  greater  than  the  affinity 
of  its  haptophore  group  for  the  complement.  A  measure  of  the 


CONCERNING  ILEMOLYSIXS.  17 

relative  affinity  is  found  in  the  degree  of  temperature  at  which 
combination  occurs.  In  the  case  of  the  lysin  obtained  by  immuniza- 
tion, which  has  already  been  described,  the  combination  of  the  blood- 
cells  with  the  corresponding  haptophore  group  of  the  immune  body 
took  place  at  0°  C. ;  the  combination  of  the  second  haptophore  group 
with  the  complement  took  place  only  at  a  higher  temperature.  At 
0°  C.  the  fluid  would  therefore  contain  immune  body  and  comple- 
ment in  a  free  state,  i.e.  uncombined.  In  this  case,  of  course,  it  is 
possible  completely  to  abstract  the  immune  body  from  this  mixture 
by  means  of  the  red  blood-cells.  This  is  the  most  favorable  case. 
Its  direct  opposite  will  be  one  in  which  the  affinity  of  the  two  hapto- 
phore groups  is  exactly  equal.  In  that  case  the  blood-cells  will 
invariably  combine  with  interbody  +  addiment  in  such  a  manner 
that  equal  amounts  of  the  two  components  are  withdrawn  from  the 
fluid.  Naturally  between  these  two  extremes  all  kinds  of  inter- 
mediate phases  may  exist  showing  variations  in  the  degree  of  affinity  of 
these  two  groups.  It  seems  to  us  that  the  most  frequent  case  is 
that  in  which  the  affinity  of  the  hsemotropic  group  of  the  interbody  is 
not  much  greater  than  that  of  the  group  fitting  the  addiment.  In 
this  case  we  are  unable  to  produce  free  addiment  by  treating  the 
mixture  with  erythrocytes;  a  certain  amount  of  interbody  always 
remains  in  the  serum  so  that  the  latter  does  not  completely  lose 
its  solvent  property.  Such  sera,  which  still  possess  solvent  property, 
cannot,  of  course,  be  used  for  experiments  in  activation. 

In  our  investigations  on  normal  sera  we  met  with  this  last  case 
surprisingly  often,  and  it  was  this  circumstance  that  made  the  study 
of  the  complements  so  difficult.  We  therefore  sought  to  find  another 
method  of  procedure,  one  by  which  these  difficulties  could  be 
avoided. 

For  analytical  purposes  it  is  essential,  as  already  stated,  to  have 
both  components  of  the  serum,  viz.,  interbody  and  complement, 
in  an  isolated  form.  The  interbody  can  at  any  tune  be  obtained 
from  the  normal  active  serum  by  heating,  but  the  production  of 
the  complement  from  the  normal  serum  is  not  entirely  successful 
because  of  the  above-mentioned  difficulties. 

We  therefore  proceeded  on  the  assumption  that  every  blood 
serum  may  contain  a  whole  series  of  different  ferment-like  bodies, 
among  which  some  would  be  capable  of  assuming  the  role  of  com- 
plement. It  was  of  course  clear  that  such  a  combination  of  circum- 
stances would  only  be  a  fortunate  chance  occurrence,  and  that  only 


18  COLLECTED  STUDIES  IN   IMMUNITY. 

by  examining  a  large  number  of  separate  cases  would  such  a  favor- 
able combination  be  found.  As  a  matter  of  fact  after  a  rather  long 
search,  we  succeeded  in  finding  such  cases. 

As  is  well  known,  dog.  serum  dissolves  guinea-pig  blood  with  great 
energy.  If  it  be  heated  to  57°  C.  it  loses  this  power,  in  accord- 
ance with  the  usual  rule.  However  if  to  the  5%  guinea-pig  blood 
mixture  some  of  this  inactive  dog-serum  is  added,  and  also  a  sufficient 
quantity  of  normal  guinea-pig  serum  (about  2  cc.  to  5  cc.  of  the  5% 
blood  mixture),  complete  solution  takes  place.  This  fact  can  be  ex- 
plained only  by  assuming  that  the  guinea-pig  serum1  contains  a 
complement  which  happens  to  fit  the  haptophore  group  of  the  inter- 
body  derived  from  the  dog,  and  that  it  thus  reactivates  this.  In 
this  case  the  proof  is  all  the  more  convincing  because  solution  is 
effected  by  the  addition  of  serum  of  the  same  species  from  which  the 
blood-cells  are  derived.  This  serum  should  be  the  best  possible 
preservative  for  the  cells,  for  it  represents  their  physiological  medium.1 

By  means  of  these  experiments  we  regard  it  as  positively  proven 
that  the  hsemolytic  action  exhibited  by  a  serum,  normally  or  in 
response  to  immunizing  procedures,  is  due,  in  the  cases  examined 
by  us,  to  the  combined  action  of  two  substances. 

Now  that  we  had  at  our  command  the  interbody  of  the  hsemolysin 
solvent  for  guinea-pig  blood,  derived  from  dog  serum,  as  well  as  a 
complement  which  reactivated  this,  we  were  ready  to  proceed  to 
the  last  of  our  demonstrations. 

To  each  of  two  test-tubes  containing  5  cc.  5%  guinea-pig  blood 
0.2  cc.  inactive  dog  serum  were  added,  after  it  had  previously  been 
ascertained  by  experiment  that  0.2  cc.  dog  serum  previous  to  heat- 
ing were  just  sufficient  completely  to  dissolve  this  amount  of  guinea- 
pig  blood.  The  mixtures  were  allowed  to  remain  at  20°  for  half  an 

1  We  succeeded  also  in  finding  other  combinations  in  which  an  analogous 
relation  in  greater  or  less  degree  could  be  demonstrated.  Of  these  we 
may  mention:  1)  guinea-pig  blood,  inactive  calf  serum,  guinea-pig  serum; 
2)  sheep  blood,  inactive  rabbit  serum,  sheep  serum;  3)  goat  blood,  inactive 
rabbit  serum,  goat  serum;  4)  guinea-pig  blood,  inactive  sheep  serum,  guinea- 
pig  serum.  The  fact  that  such  an  interbody,  i.e.,  one  derived  from  one 
animal  species,  finds  fitting  complements  not  only  in  its  own  serum  but 
also  in  that  of  different  species,  is  of  considerable  importance  in  the  question 
whether  curative  sera  can  be  made  harmless  to  man  by  means  of  pasteurization. 
Possibly  this  would  serve  to  explain  why  heating  of  the  diphtheria  curative 
serum,  introduced  by  Spronck,  has  not  realized  the  expectations  a  priori  held 
out  for  the  procedure. 


COXCERXIXG  H.EMOLYSINS.  19 

hour  and  then  centrifuged.  The  sediments  thus  obtained  were 
washed  with  salt  solution  and  again  centrifuged.  If  now  to  one 
of  these  sediments  physiological  salt  solution  was  added,  and  to 
the  other  1.5  cc.  guinea-pig  serum,  complete  solution  resulted  in  the 
latter,  while  the  former  remained  undissolved.  This  proves  that 
the  interbody  was  completely  anchored  by  the  blood-corpuscles. 
The  fluid  obtained  by  centrifuging  did  not  dissolve  guinea-pig 
blood,  even  when  considerable  guinea-pig  serum  was  added.  It 
did  not,  therefore,  contain  any  free  interbody  derived  from  the  dog 
serum  first  added. 

By  these  experiments  we  became  convinced  that  ha?molysis  in 
general  is  due,  not  to  a  simple  body,  but  to  the  combined  action  of 
two  distinct  substances.  At  the  present  time  we  have  no  general 
method  to  demonstrate  this  for  each  individual  case,  and  the  solution 
of  the  problem  therefore  is  now  possible  only  under  either  of  the 
above-mentioned  favorable  conditions:  •(!)  when  the  two  hap- 
tophore  groups  of  the  interbody  differ  greatly  in  their  affinity;  and 
(2)  when,  by  means  of  a  combination  whose  discovery  depends  on 
chance,  an  activating  complement  is  found.  Where  these  conditions 
are  not  fulfilled,  the  solution  of  the  problem,  for  the  present  at  least, 
is  impossible.  This,  for  example,  is  the  case  with  ichthyotoxin,  the 
hsemolytlc  constituent  of  eel  serum.  It  is  extremely  easy  to  inactivate 
this  eel  serum,  slight  warming  for  fifteen  minutes  to  54°  C.  sufficing, 
but  thus  far  we  have  been  entirely  unsuccessful  in  reactivating  it, 
because  we  have  been  unable  to  find  the  requisite  complement. 

Considering  their  multiplicity,  it  is  but  natural  that  we  are  only 
just  getting  a  deeper  insight  into  the  nature  of  the  substances  in 
normal  blood  serum.  It  is  obvious  also  that  a  great  many  questions 
whose  solution  is  of  importance  present  themselves,  especially  in 
connection  with  the  substances  discussed  by  us. 

The  first  question  to  be  considered  is  that  of  the  multiplicity  of 
the  haemolysins  contained  in  a  given  normal  serum.  According  to 
our  observations  it  is  very  probable  that  the  ability  of  serum  of  one 
species  to  dissolve  the  blood-cells  of  various  other  species  is  de- 
pendent on  the  action,  not  of  a  single  lysin,  but  of  several  lysins. 
If,  for  example,  dog  serum  dissolves  the  blood-cells  of  guinea-pigs 
and  of  rabbits,  it  must  be  assumed  that  a  multiplicity  of  interbodies 
and  of  corresponding  complements  effects  this  action.  Some  of  the 
ways  in  which  the  solution  of  this  problem  can  be  approached  are 
as  follows: 


20  COLLECTED  STUDIES  IN  IMMUNITY. 

(1)  The  isolated  destruction  of  single  lysins  by  means  of  thermic 
and  chemic  influences. 

(2)  The  binding  of  the  different  lysins  by  means  of  corresponding 
species  of  blood,  thus  making  their  elective  removal  possible.     With 
red  blood-cells  this  procedure,  to  which  we  shall  return  in  a  sub- 
sequent  article,   offers   many   technical   difficulties.     On  the  other 
hand,  with  a  different  kind  of  specific  constituent  of  the  serum, 
namely,  the  agglutinins,  this  method  is  easily  applied,  as  can  be  seen 
by  the  experiments  of  Bordet l  made  in  connection  with  our  first 
.experiments  and  carried  out  by  the  methods  employed  by  us. 

(3)  A  separation  of  the  lysins  also  seems  possible  through  im- 
munization, by  means  of  which  one  is  able  to  obtain  antibodies 
against  the  normal  lysins.     Thus  Kossel,  Camus,  and  Gley,  by  treat- 
ing animals  with  the  strongly  globulicidal  eel  serum,  have  obtained 
a  serum  which  neutralizes  the  action  of  this  eel  serum,  in  other  words, 
one  containing  an  antilysin.     Evidently  this  reactively  formed  anti- 
body thrusts  itself  into  the  hsemotropic  group  of  the  interbody  and 
thus  deflects  this  from  the  erythrocyte.    Our  attempts,  based  on 
these  premises,  to   produce   an  isolated  antibody  for  some  of  the 
lysins  have   thus  far  been  unsuccessful.     Thus    a    serum    derived 
from  rabbits  after  these  had  been  treated  with  goat  serum,  protected 
the  rabbit  erythrocytes  against  solution  by  goat  serum.     At  the  same 
time,  however,  it  protected  the  blood  of  guinea-pigs  and  rats  against 
the  same  influence,  and  even  prevented  the  hsemolytic  action  of  dog 
serum   on  rabbit  blood.     From   this   fact   we   must   conclude   that 
immunization  with  one  serum  produces  a  whole  series  of  different 
antilysins.     Clearly  this  is  to  be  explained  by  assuming  that  a  serum 
contains  a  great  number  of  different  complexes  possessing  haptophore 
groups,  of  which  many,  whether  they  are  toxic  or  not,  are  able  to 
excite  the  production  of  corresponding  antibodies. 

This  surprising  multiplicity  of  substances,  present  in  the  blood, 
which  possess  haptophore  groups  (hsemolysins,  agglutinins,  ferments, 
antiferments)  is  very  readily  harmonized  with  Ehrlich's  views. 
According  to  his  conception  all  these  substances  represent  side- 
chains  of  the  protoplasm,  which  have  been  thrust  off  and  have  reached 
the  circulation.  The  physiological  object  of  the  side-chains  is,  as 
Ehrlich  stated  in  1885,2  to  bind  assimilable  substances  to  the 
protoplasm  so  that  these  may  serve  as  nutriment  for  the  latter. 

1  Inst.  Pasteur,  March  1899. 

3  Ehrlich,  Sauerstoffbediirfniss  des  Organismus.     Berlin,  1885. 


CONCERNING  H.EMOLYSINS.  21 

A  large  part  of  these  side-chains  may,  under  suitable  circumstances, 
be  thrust  off  and  thus  appear  in  the  blood. 

Considering  the  large  number  of  organs  in  the  body  and  the  mani- 
fold chemistry  of  their  protoplasm,  it  should  not  surprise  us  that 
the  blood,  which  represents  all  the  tissues,  can  be  filled  with  innumer- 
able side-chains;  and  it  is  not  at  all  astonishing,  considering  the 
constantly  changing  chemistry  of  the  organism  (influenced  by  a  large 
number  of  factors  such  as  race,  sex,  nutrition,  labor,  secretion,  con- 
ditions of  the  surrounding  medium,  etc.)  that  the  serum  should  be 
subject  to  constant  qualitative  fluctuations.  Such  variations  are 
seen  in  the  examples  already  mentioned,  showing  the  behavior  of 
sera  of  normal  animals.  Goat  serum  at  one  time  possesses  a  slight 
solvent  action  on  sheep  blood,  at  other  times  this  is  entirely  absent. 
Dog  serum  in  one  case  dissolves  the  red  cells  of  cats  very  strongly, 
in  another  case  it  does  not  do  so  at  all.  The  action  of  rabbit  serum 
on  guinea-pig  blood  shows  a  special  variability. 

A  very  interesting  example  is  afforded  by  lamprey  serum,  which, 
as  is  well  known,  possesses  an  extraordinarily  toxic  action  for  labora- 
tory animals  in  general  and  also  for  red  blood-cells  in  vitro.  Dr. 
Schonlein  of  Naples,  whose  recent  death  we  lament,  was  kind  enough 
to  experiment  with  this  for  us.  His  investigations  showed  that  the 
serum  of  a  not  inconsiderable  number  of  lampreys  possesses  no 
toxic  action  at  all,  so  that  it  could  be  injected  into  rabbits  intra- 
venously in  amounts  of  2  cc.  without  any  damage  whatever. 

It  is  clear  that  this  extensive  variability  enormously  increases  the 
difficulties  in  investigating  these  sera.  Thus  on  repeating  the  well- 
known  experiment  of  Buchner,  whereby  a  mixture,  in  certain  pro- 
portions, of  dog  and  rabbit  sera  loses  its  haemolytic  property  for 
guinea-pigs  in  the  course  of  twenty-four  hours,  we  were  able  to  com- 
pletely confirm  Buchner's  results  in  three  cases,  while  in  five  other 
cases  the  haemolytic  effect  was  only  more  or  less  lost. 

We  believe  that  all  these  investigations  support  the  view  we  have 
already  expressed  regarding  the  nature  of  the  complex  poisons  of  the 
blood-sera,  v.  Dungern  (Muench.  med.  Wochenschr.,  1899,  No.  14), 
basing  his  action  on  some  new  experiments  of  his,  has  accepted  our 
views.  We  can  content  ourselves,  therefore,  with  merely  mentioning 
another  view,  recently  expressed  by  Bordet l  He  has  confirmed  the 
statements  made  by  us  regarding  the  fixation  of  the  specific  immune 
body  by  means  of  the  corresponding  erythrocyte,  and  he  has  ad- 
1  Annal.  de  1'Instit.  Pasteur,  April  1899. 


22  COLLECTED  STUDIES  IN  IMMUNITY. 

mitted  that  the  fixation  process  is  connected  with  the  solvent  process, 
but  he  believes  that  the  nature  of  this  connection  requires  a  special 
hypothesis : 

"On  pourrait  rapprocher,  si  une  comparaison  un  peu  grossiere 
etait  permise,  la  modification  apportee  par  la  substance  sensibila- 
trice  [our  immune  body]  sur  le  globule,  de  celle  qui  consisterait  a 
changer  la  structure  d'une  serrure,  de  fagon  a  y  permettre  Tintroduc- 
tion  facile  d'une  ou  de  plusieurs  clefs  qui  n'y  entraient  pas  auparavant 
ou  nV  penetraient  qu'avec  difficulte.  Deux  clefs  suffisamment  sem- 
blables  enteront  des  lors  indiff element." 

One  could  therefore  picture  the  mode  of  action  of  the  two  sub- 
stances as  it  is  conceived  by  Bordet  to  be  like  a  safety  lock  which  re- 
quires two  keys  to  open  it,  of  which  the  first  is  necessary  in  order 
to  make  the  main  lock  accessible. 

Against  this  mechanical  conception  it  can  be  urged  that  the  keys 
do  not  fly  into  the  lock  of  their  own  accord,  but  that  certain  forces 
are  necessary  to  effect  this.  Our  theory  supplies  a  very  simple 
explanation  for  this ;  the  driving  force  is  the  chemical  affinity  between 
the  fitting  groups.  The  entire  line  of  experiments  made  by  us  was 
designed  to  show  whether  the  two  substances,  together,  combined 
with  the  blood-cells  at  one  place  or  whether,  separately,  at  two  different 
places.  Our  decision  was  determined  by  the  demonstration  that 
the  addiment  was  in  no  way  fixed  by  the  red  blood-cells.  Had 
Bordet  repeated  not  only  one  of  our  experiments,  but  the  entire 
series,  the  inapplicability  of  his  hypothesis  would  have  become  evi- 
dent to  him. 

If  active  immune  serum  is  treated  with  red  blood-cells,  at  0°  C. 
as  described  in  our  first  article,  thus  fixing  the  immune  body,  the 
lock,  according  to  Bordet,  is  made  accessible,  i.e.  the  conditions 
are  fulfilled  whereby  the  addiment  (Bordet's  alexin)  could  pene- 
trate to  the  blood-cells.  As  a  matter  of  fact,  however,  under  these 
circumstances  the  addiment  does  not  do  so.  This,  as  well  as  the 
new  facts  mentioned  in  the  present  article,  harmonize  best  with 
our  theory. 

If,  however,  this  mode  of  action  of  the  lysins  is  accepted,  it  will 
be  impossible  not  to  hold  the  same  views  regarding  the  living  pro- 
toplasm, and  assume  in  this  the  presence  of  side-chains  of  peculiar 
character  which  are  designed  to  grasp  highly  complicated  substances. 
It  must  further  be  assumed  that  these  side-chains,  beside  their  grasp- 
ing group,  are  endowed  with  a  second  group  which,  by  fixation  of 
peculiar  ferments,  effects  a  digestive  action. 


III.  STUDIES   ON  HAEMOLYSIS.1 

THIRD  COMMUNICATION.2 
By  Professor  Dr.  P.  EHRLICH  and  Dr.  J.  MORGENROTH. 

BY  injecting  one  animal  with  the  cells  of  another,  we  can  produce 
substances  in  the  serum  of  the  first,  which  have  a  specific  damaging 
or  destructive  influence  on  these  cells.  This  possibility  has  within 
a  short  time  extended  the  theoretical  doctrines  of  immunity  in  vari- 
ous directions.  First  Belfanti  and  Carbone  showed  that  the  serum 
of  animals,  after  these  had  been  treated  with  blood-cells  of  a  differ- 
ent species,  acquires  a  high  degree  of  toxicity  for  just  this  species- 
Shortly  afterward,  Bordet  was  able  to  demonstrate  that  this  toxicity 
in  corpore  corresponds  to  a  specific  haemolysis  in  vitro.  This  was 
confirmed  independently  by  von  Dungeni  and  Landsteiner  by  experi. 
ments  published  somewhat  later,  and  further  by  those  of  our  own 
mentioned  in  previous  communications.  The  result  of  the  experi- 
ments is  always,  that,  following  the  introduction  of  red  blood-cells 
of  one  species  into  the  organism  of  another,  a  hsernolysin  is  formed 
which  so  injures  the  blood-cells  of  the  first  species  that  their  haemo- 
globin goes  into  solution.  Bordet  also  showed  that  this  haemolysis 
depends  on  the  action  of  two  substances  in  the  haemolytic  serum. 

The  importance  of  this  subject,  due  specially  to  the  complete 
analogy  between  the  hsemolytic  and  the  bacteriolytic  processes, 
led  us  to  a  detailed  study  of  the  mechanism  of  these  processes.  We 
were  able  to  show  that  the  substance  produced  by  immunization,  the 
immune  body,  possesses  a  maximum  chemical  affinity  for  the  corre- 
sponding blood-cell.  This  affinity  is  due  to  the  presence  of  a  specific 
combining  group  in  the  molecule  of  the  immune  body,  which  fits 
to  a  corresponding  group  in  the  protoplasm  of  the  erythrocyte. 
Beside  this,  the  immune  body  possesses  a  second  combining  group 


1  Reprint  from  the  Berliner  klin.  Wochenschr.  1900,  No.  21. 

2  See  pages  1  and  11. 


23 


24  COLLECTED  STUDIES  IN  IMMUNITY. 

which  fits  to  a  group  in  a  ferment-like  body  of  normal  serum,  namely, 
the  complement  (addiment).  By  virtue  of  these  two  haptophore 
groups,  the  immune  body  functionates  as  a  coupler  or  interbody. 
carrying  the  action  of  the  complement  over  onto  the  red  blood-cells, 
In  order  to  facilitate  expression,  that  combining  group  of  the  pro- 
toplasmic molecule  to  which  the  introduced  group  is  anchored  will  here- 
after be  termed  receptor.  The  side-chain,  for  example,  which  com- 
bines with  the  tetanus  toxin  in  the  organism  is  such  a  receptor.  The 
tetanus  antitoxin  itself  is  nothing  but  the  surplus  of  receptors  thrust 
off  into  the  blood.  Similarly,  that  complex  which  later  functionates 
as  immune  body  is  a  receptor  before  being  thrust  off. 

In  the  further  course  of  these  investigations  it  has  been  found 
that  the  function  to  produce  peculiar  antibodies  analogous  to  immune 
bodies  is  not  confined  to  bacteria  and  erythrocytes.  Cells  of  the 
most  varied  kind,  provided  they  are  absorbed,  excite  the  production 
of  immune  bodies,  in  conformity  with  the  requirements  of  the  side- 
chain  theory.  Landsteiner,  Metchnikoff,  and  Moxter  succeeded 
in  producing  an  immune  serum  against  spermatozoa;  von  Dungern, 
a  specific  serum  which  acted  on  ciliated  epithelium;  and  Mecthni- 
koff,  an  immune  serum  against  leucocytes  and  kidney  epithelium. 
Here  also  in  the  cases  examined  for  this  purpose  (v.  Dungern,  Moxter) 
it  could  be  shown  that  the  specific  active  substances  are  of  complex 
nature,  consisting  of  an  immune  body  and  a  corresponding  comple- 
ment, and  that  the  immune  body  possesses  a  specific  affinity  for  the 
corresponding  cells. 

The  great  theoretical  significance  of  these  investigations  which 
open  up  a  new  field  to  the  study  of  immunity  is  clearly  apparent, 
but  whether  in  the  near  future  they  will  have  any  practical  results 
remains  to  be  seen. 

In  the  pursuit  of  these  studies,  we  were  led  to  extend  our  researches 
into  another  direction  which  seemed  to  us  of  special  importance 
in  the  understanding  of  pathological  processes. 

The  experimental  investigations  thus  far  made  have  dealt  exclu- 
sively with  the  changes  in  the  serum  which  occur  when  an  animal 
is  made  to  absorb  foreign  cell  material.  This  mode  of  experiment, 
however,  is  not  limited  in  any  way  by  the  nature  of  the  subject,  but 
is  dependent  entirely  on  the  will  of  the  experimenter,  and  it  there- 
fore lacks  all  physiological  analogy. 

In  pathology,  the  changes  foremost  to  be  considered  are  those 
resulting  from  the  absorption,  by  an  organism,  of  its  own  cell  mate- 


STUDIES  ON   ILEMOLYSIS.  25 

rial.  Such  occasions  are  presented  by  many  different  diseases. 
Keeping  to  the  blood,  for  example,  if  an  individual  suffers  a  con- 
siderable subcutaneous  hemorrhage  or  one  into  a  body-cavity,  or 
if  part  of  his  blood-corpuscles  are  destroyed  and  dissolved  by  certain 
blood-poisons,  the  essential  conditions,  just  as  in  an  experiment, 
are  given  for  the  reactive  formation  of  substances  possessing  specific 
injurious  affinities  for  these  blood-cells.  The  same,  however,  can 
apply  to  other  tissues;  for  every  acute  atrophy  of  an  organ's  paren- 
chyma can  lead  to  the  absorption  of  cell  material  and  to  its  conse- 
quences. The  conditions  necessary  for  the  development  of  specific 
cell  poisons  may  be  presented  by  various  circumstances,  thus,  when, 
spontaneously  or  under  the  influence  of  arsenic,  large  lymph-gland 
tumors  are  absorbed;  when  a  struma  melts  and  disappears  under 
specific  treatmnt;  when  the  white  blood-cells,  owing  to  the  action 
of  toxins  or  other  substances,  are  caused  to  disintegrate;  when, 
owing  to  certain  metabolic  or  infectious  diseases,  acute  atrophy  of 
the  liver  ensues,  etc.  We  shall  further  have  to  assume  that  these 
conditions  can  be  fulfilled,  in  a  wider  sense,  when,  under  the  influence 
of  certain  general  diseases,  there  occurs  active  dissolution  of  or- 
ganized material  of  any  kind  instead  of  atrophy  of  a  single  organ. 

It  is  therefore  of  the  highest  pathological  importance  to  determine 
whether  the  absorption  of  its  own  body  material  can  excite  reactive 
changes  in  the  organism,  and  what  the  nature  of  these  changes  is.  The 
simplest  conditions  and  those  most  accessible  to  experimental  study  are 
those  which  arise  on  the  absorption  of  blood-cells.  But  here  we  face  a 
curious  dilemma.  If  an  animal  organism,  when  injected  with  blood- 
cells  of  foreign  species,  always  produces  a  specific  haBmolysin  for 
each  of  these  species,  it  must  surely  be  following  a  natural  law;  and 
it  is  improbable  that  this  law  w^hich  applies  in  any  particular  number 
of  cases  should  be  suspended  in  the  case  of  blood-cells  of  the  same 
individual.  On  the  other  hand,  it  is  not  to  be  denied  that  the  forma- 
tion of  such  hsemolytic  substances  would  appear  dysteological  in  the 
highest  degree.  For  example,  if,  in  an  individual  who  has  had  an 
extensive  haemorrhage  into  a  body-cavity,  the  absorption  of  this 
blood  caused  the  formation  of  a  blood  poison  which  destroyed  the 
rest  of  the  blood-cells,  this  would  be  a  phenomenon  whose  actual 
occurrence  lacks  any  clinical  evidence  whatever  and  one  which  no  one 
is  willing  to  accept. 

It  cannot  be  doubted  that  the  organism  seeks  a  way  out  of  this* 
difficulty  by  means  of  certain  regulating  contrivances,  whose  deter- 


26  COLLECTED  STUDIES   IN  IMMUNITY. 

mination  will  be  of  the  highest  interest.  To  be  sure  the  study  of 
this  question  offers  considerable  difficulties,  difficulties  through 
which  previous  experiments  in  this  direction  have  been  brought 
to  naught.  (Belfanti  and  Carbone,  Bordet.) 

We  have  from  the  beginning  maintained  that  it  is  possible  to 
gain  an  insight  into  these  processes,  only  when  any  changes  occurring 
in  the  serum  are  determined  by  means  of  frequent  and  progressive 
examinations.  Small  laboratory  animals,  because  of  the  amount 
of  blood  required  for  these  continuous  examinations,  are  therefore 
unavailable,  and  hence  we  selected  goats  as  being  best  adapted  for 
these  experiments. 

After  it  had  been  determined  that  a  single  injection  of  a  large 
amount  of  blood  sufficed  to  produce  the  specific  hasmolytic  sub- 
stances in  the  serum,  we  usually  injected  our  animals  once  with  a 
large  amount  of  goat-blood.  (800-900  cc.  for  a  goat  of  35-40  kg.) 
In  order  to  overwhelm  the  body  as  rapidly  as  possible  with  the  con- 
stituents of  the  blood-cells,  we  made  use  of  intraperitoneal  injections. 
For  .the  same  reason  we  thought  it  best  not  to  inject  intact  blood- 
corpuscles,  but  to  inject  blood  which  had  been  made  laky  by  the 
addition  of  water.  We  argued  that  blood-cells  of  the  same  species 
as  the  animal  injected  would  be  destroyed  very  slowly  in  the  peri- 
toneal cavity  of  this  animal,  and  that  consequently  the  absorption 
would  be  so  gradual  as  to  prevent  the  occurrence  of  what  may  be 
termed  an  "  ictus  immunisatorius."  From  the  second  or  third 
day  on,  we  withdrew  samples  of  serum  from  the  animals  so  treated, 
and  tested  the  solvent  action  on  the  blood  of  numerous  other  goats. 
Our  method  generally  was  first  to  determine  whether  any  indica- 
tions of  hsemolytic  action  were  •  present.  For  this  purpose  a  drop 
of  normal  goat  blood  was  allowed  to  fall  into  undiluted  serum  of 
the  treated  goats,  and  the  occurrence  of  any  red  coloration  looked 
for.  If  this  test  was  positive,  we  proceeded  to  test  the  hsemolysin 
in  the  usual  manner  by  adding  decreasing  amounts  of  this  serum 
to  tubes  containing  1  cc.  of  a  5%  mixture  of  goat-blood  in  0.85% 
salt  solution. 

.  With  these  preliminary  remarks  we  proceed  to  our  first  posi- 
tive test  (February  16,  1900).  The  subject  of  this  was  a  strong 
male  goat,  buck  A,  weighing  33.5  kg.,  into  whom  there  were  injected 
intraperitoneally  920  cc.  goat-blood  (mixed  from  the  blood  of  goats 
1,  2,  and  3)  made  laky  by  the  addition  of  750  cc.  water.  From  the 
second  day  on,  small  amounts  of  blood  were  withdrawn  daily  for 


STUDIES  ON  HAEMOLYSIS.  27 

the  purpose  of  obtaining  serum.  This  serum,  as  we  had  antici- 
pated, never  showed  a  trace  of  haemoglobin  coloration.  As  early 
as  the  second  day,  a  slight  solvent  action  for  the  blood  of  goats  4  and 
5  was  developed.  A  drop  of  the  blood  allowed  to  fall  into  the  undi- 
luted serum  of  buck  A  suffered  partial  solution,  so  that  after  the 
blood-corpuscles  had  sedimented,  the  serum  remained  slightly  tinged 
with  red.  By  the  fifth  day  the  solvent  property  had  increased 
considerably;  0.5  cc.  serum  completely  dissolving  1.0  cc.  of  the 
5TC  blood-mixture  of  goat  No.  4.  By  the  seventh  day  the  action 
had  reached  its  maximum.  0.3  cc.  serum  produced  complete  solu- 
tion (Xo.  4);  0.07  a  just  appreciable  effect. 

As  we  now  had  at  our  disposal  a  sufficient  amount  of  haemolysin. 
we  sought  to  determine  whether  this  haemolysin  dissolved  all  goat 
blood-corpuscles  without  exception.  We  found  that  of  nine  goats 
which  we  examined,  the  majority  wrere  markedly  sensitive  to  this 
hsemolysin.  Thus  goats  Nos.  1,  2,  4,  5,  6,  and  9  were  highly  sen- 
sitive; two  goats,  Nos.  3  and  8,  somewhat  less  so;  and  only  one, 
No.  7,  (which  had  previously  been  treated  for  some  time  with  the 
expressed  juice  of  eel  muscle,)  showed  so  slight  a  susceptibility  that 
even  undiluted  serum  failed  to  cause  strong  solution. 

After  noting  these  results  it  was  important  to  determine  the 
behavior  of  the  blood-cells  of  this  buck  toward  the  haemolysin  of 
his  own  serum.  If  a  drop  of  blood  was  added  to  the  serum,  in  vitro, 
not  even  a  trace  of  solution  occurred.  These  blood-cells  then  were 
entirely  insusceptible  to  the  haemolysin  of  their  own  serum,  as  had 
already  been  indicated  by  the  absence  of  haemoglobin  coloration 
in  the  freshly  drawn  serum. 

If  we  designate  the  specific  haemolysin  developed  by  the  injec- 
tion of  blood  of  foreign  species  as  heterolysin,  then  we  must  designate 
the  haemolysin  due  to  the  injection  of  blood  of  the  same  species  as 
isolysin.  In  no  case,  however,  and  this  is  to  be  emphasized,  are 
we  here  dealing  with  an  autolysin,  i.e.  a  lysin  which  dissolves  the 
blood-cells  of  the  animal  in  whose  serum  it  circulates.  However, 
such  a  condition  is  not  at  all  a  matter  of  course,  and  the  question 
arises  why  the  isolysin  in  this  case  does  not  also  functionate  asauto- 
lysin. 

The  toxins  as  well  as  the  haemolysins  can  act  only  when  they 
are  anchored  by  certain  haptophore  groups,  the  receptors,  whereby 
the  action  of  the  poisons  is  concentrated  on  the  cells  possessing  these 
receptors.  If  these  groups  are  lacking,  the  poison  has  no  point  of 


28  COLLECTED  STUDIES  IX  IMMUNITY. 

attack.  We  have  already  demonstrated  that  a  hsemolysin,  or  rather 
its  immune  body,  is  anchored  by  the  erythrocytes,  and  the  solution 
of  the  above  question  therefore  becomes  very  easy.  To  begin,  we 
have  determined  that  the  isolysin  behaves  like  a  typical  hsemolysin 
of  the  well-known  kind.  It  loses  its  action  by  being  heated  for 
half  an  hour  to  55°  C.  (destruction  of  the  complement)  and  is  reac- 
tivated by  the  addition  of  a  corresponding  amount  of  normal  goat 
serum. 

Next  we  have  determined  that  the  immune  body  of  the  isolysin 
is  bound  by  the  susceptible  blood-cells  in  typical  fashion;  that  the 
blood-cells  of  the  immunized  animal,  however,  take  up  only  traces 
of  the  immune  body  in  vitro,  amounts  far  less  than  those  taken  up 
by  the  almost  insensitive  blood-cells  of  goat  No.  7.  This  phenomenon 
can  at  once  be  ascribed  to  a  slight  mechanical  absorption.  We  see, 
therefore,  that  the  serum's  own  insensitive  blood-cells  are  incapable 
of  anchoring  the  specific  immune  body  of  the  isolysin. 

This  result  can  be  explained  in  either  of  two  ways.  It  may  be 
assumed  that  the  blood-cells  lack  this  receptor  entirely,  or  that, 
although  the  cells  possess  the  receptor,  the  affinity  of  this  had  already 
been  satisfied  by  the  immune  body  in  the  circulation.  In  the  latter 
case,  however,  it  is  incomprehensible  why  the  blood-cells  were  not 
dissolved  by  the  complement  also  circulating  in  the  blood.  Further 
reasons  against  the  latter  assumption  will  be  apparent  later,  and 
so  we  shall  at  once  discuss  a  series  of  facts  which,  according  to  our 
views,  demonstrate  that  the  insusceptibility  of  the  blood-cells  in 
this  case  is  due  to  an  absolute  lack  of  these  receptors. 

Assuming  that  a  given  toxin,  in  an  organism,  finds  receptors 
which  anchor  it,  the  injection  of  this  toxin  will  be  followed  by  the 
production  of  a  corresponding  antibody.  If,  however,  an  organism 
lack  receptors  for  this  poison,  the  first  essential  for  the  production 
of  an  antibody  will  be  wanting.  In  the  development  or  non-develop- 
ment of  antibodies  we  shall  have  an  indication  of  the  presence  or 
absence  of  receptors. 

Now,  the  hsemolysins  belong  to  the  class  of  poisons  which  pro- 
duce antibodies.  We  ourselves  have  demonstrated  that  the  normal 
haemolysins  of  dog's  and  goat's  serum,  when  injected  into  a  foreign 
animal  body,  excite  the  production  of  antihsemolysins.  The  ques- 
tion was  whether  the  isolysin  when  injected  into  the  organism  of 
other  goats  would  be  able  to  cause  the  production  of  an  anti-isolysin. 
In  order  to  save  material  we  injected  a  young  goat  (No.  10),  whose 


STUDIES  ON  HAEMOLYSIS.  29 

Hood-cells  we  had  previously  shown  to  be  very  sensitive  to  the  iso- 
lysin,  several  times  with  considerable  quantities  of  serum  A.  As 
a  matter  of  fact  an  antibody  was  developed,  so  that  0.4  cc.  of  the 
serum  thus  obtained  were  able  to  protect  1  cc.  of  a  5%  sensitive 
goat-blood-cell  mixture  against  solution  by  isolysin  A  (0.5  cc.).  The 
blood-cells  of  this  same  goat  No.  10,  on  the  contrary,  after  they  had 
been  repeatedly  washed  with  physiological  salt  solution  to  free  them 
from  serum,  proved  just  as  susceptible  to  the  isolysin  as  before. 
Hence  it  follows  that  the  isolysin  here  concerned,  isolysin  A,  causes 
the  production  of  antilysins  in  the  body  of  the  same  species  when 
it  finds  fitting  receptors. 

From  this  we  conclude  that  the  insensitiveness  of  the  red  blood-cells 
can  only  be  due  to  the  lack  of  receptors  for  the  isolysin.  A  further 
conclusion  must  be  that  these  receptors  are  not  present  hi  any  other 
tissue  of  buck  A,  that  they  are  absent  in  the  entire  organism,  for  other- 
wise there  should  have  been  a  formation  of  anti-isolysin. 

It  goes  without  saying  that  we  repeated  these  experiments  on 
a  large  number  of  animals  in  order  to  exclude  all  accidental  phenom- 
ena. In  the  course  of  these  experiments  we  noted  numerous  and 
interesting  variations  in  the  reaction  to  isolysins. 

Of  special  interest  is  goat  B,  which  had  been  treated  exactly  like 
buck  A.  At  first  it  seemed  as  though  the  experiment  with  this 
animal  would  run  an  entirely  different  course,  for  during  the  first 
fourteen  days  we  were  unable  to  detect  even  a  suggestion  of  an  iso- 
lysin. The  red  cells,  however,  remained  completely  sensitive  to  the 
isolysin  derived  from  buck  A.  Then  suddenly  on  the  fifteenth  day 
after  the  blood  injection  a  hsemolysin  made  its  appearance,  one 
which  acted  on  goat  blood  quite  as  strongly  as  the  isolysin  of  buck 

A.  The  animal's  own  blood-cells  were  just  as  insensitive  to  this 
haemolysin  as  were  those  in  the  first  experiment  to  theirs.     Here  also, 
then,  we  were  dealing  with  an  isolysin,  not  an  autolysin.     The  sen- 
sitiveness   of    the    blood    toward    isolysin   A    continued.     We    now 
examined  the  majority  of  our  goats  hi  order  to  determine  their  sen- 
sitiveness to  this  isolysin,  and  found  that  some  animals  which  were 
highly  sensitive  to  isolysin  A  were  very  slightly  sensitive  to  isolysin 

B,  and  vice  versa.     The  blood  of  buck  A  occupied  a  peculiar  place. 
It  was  as  completely  insensitive  to  isolysin  B  as  it  was  to  that  of 
its  own  serum. 

From  the  behavior  of  the  blood  of  the  various  animals  toward 
these  two  isolysins.  it  was  clear  that  these  isolysins  were  essentially 


30  COLLECTED  STUDIES  IN  IMMUNITY. 

different.  This  was  positively  proven  by  the  fact  that  the  anti- 
isolysin  A  was  entirely  ineffectual  against  isolysin  B.  The  difference 
between  these  two  isolysins  is  further  illustrated  by  the  difference 
of  the  intervals  between  blood  injection  and  isolysin  formation.  In 
the  one  case  this  was  only  a  few  days  and  in  the  other  fourteen  days. 
That  the  injection  of  the  goat  blood  should  result  in  the  formation 
of  two  entirely  distinct  and  easily  differentiated  icolysins  was  cer- 
tainly a  remarkable  phenomenon.  And  yet  this  did  not  exhaust 
the  multiplicity  of  the  isolysins. 

In  a  third  goat,  C,  (injected  on  the  same  day  as  B  and  with  sim- 
ilar amounts  of  the  same  blood,)  a  haemolysin  C  appeared  on  the 
seventh  day  which  again  differed  from  isolysins  A  and  B.  This, 
furthermore,  proved  itself  an  isolysin,  for  the  blood-cells  of  the  ani- 
mal were  entirely  insensitive  to  its  action,  though  they  were  sensitive 
to  isolysins  A  and  B.  This  fact  shows  that  isolysin  C  differed  from 
isolysins  A  and  B.  It  is  specially  noteworthy  that,  although  the  two 
goats  B  and  C  were  injected  at  the  same  time  with  similar  amounts 
of  the  same  blood,  they  should  develop  different  isolysins.  This 
observation  is  particularly  important  because  it  shows  that  the 
constitution  of  the  isolysin  is  dependent  on  the  individuality  of  the 
animal  in  which  it  is  developed. 

It  is  also  very  remarkable  that  these  three  isolysins,  A,  B,  and  C, 
were  able  to  destroy  not  only  goat  blood-cells,  but  also  those  of 
sheep.  The  sheep  erythrocytes  therefore  possess  three  different 
groups  which  are  identical  with  those  of  these  goat  blood-cells,  or 
at  least  are  closely  related  to  them.  On  the  other  hand  still  another 
isolysin,  D,  does  not  dissolve  sheep  blood-cells. 

After  having  observed  three  different  isolysins  in  three  different 
goats,  we  are  in  no  wise  to  assume  that  this  exhausts  the  possibilities.1 
On  the  contrary,  it  seems  highly  probable  that  by  further  experi- 
ments we  shall  come  to  know  other  isolysins.  Nevertheless  it  must 
not  be  assumed  that  this  variation  of  the  isolysins  is  unlimited. 
It  is  to  be  expected  that  a  sufficient  repetition  of  the  experiments 
will  finally  lead  us  to  recognize  a  certain  cycle  of  constantly  repeat- 
ing types.  The  attainment  of  this  goal,  however,  is  rendered  very 

1  Note  on  revision. — In  the  mean  time  we  have  obtained  a  fourth  isolysin, 
D,  which  differs  from  isolysins  B  and  C  in  the  fact  that  it  dissolves  the  blood- 
cells  of  B  and  C.  Erythrocytes  of  A  are  not  dissolved,  but  the  isolysin  differs 
from  A  in  its  behavior  to  various  normal  kinds  of  goat  blood.  The  behavior 
of  isolysin  D  toward  sheep  blood  has  already  been  mentioned. 


STUDIES  ON  HAEMOLYSIS.  ,    31 

tedious  by  the  fact  that  in  some  cases  in  which  the  production  cf 
an  isolysin  is  attempted  after  the  method  already  outlined,  no  iso- 
lysin  is  formed.  We  have  records  of  a  number  of  goats  hi  which 
the  injection  of  goat  blood  produced  apparently  no  effect  whatever; 
among  these  is  one  which  was  injected  with  its  own  blood. 

The  difference  in  the  isolysins  in  their  dependence  on  the  injected 
blood  and  on  the  individuality  of  the  treated  animal,  the  fact  that 
there  is  formed  always  an  isolysin,  not  an  autolysin,  the  special  con- 
ditions governing  the  formation  of  the  anti-isolysins,  the  failure  of 
the  isolysin  reaction  in  certain  cases, — all  these  make  the  problems 
connected  with  the  above  facts  appear  very  complicated,  and  make 
it  necessary  now  to  analyze  these  more  closely. 

Every  red  blood-cell  possesses  a  large  number  of  side-chains  with 
haptophore  groups,  each  of  which  is  able  to  combine  in  the  animal 
body  with  fitting  receptors.  Let  us,  in  our  own  case,  designate  such 
a  group  of  the  injected  goat  erythrocytes  as  group  a,  and  a  corre- 
sponding receptor  as  receptor  a.  There  will  then  be  presented  two 
possibilities.  First  is  the  possibility  that  the  a  receptor  is  entirely 
absent  in  the  organism  of  the  goat  into  which  the  blood  is  injected. 
If  this  be  the  case,  there  is  lacking  the  essential  condition  for  the 
formation  of  any  reactive  product,  and  the  result  of  the  injection 
will  be  entirely  negative. 

If,  however,  the  second  possibility  obtains,  and  a  receptors  are 
present  in  the  body  of  the  animal  injected,  there  are  again  two  ways 
in  which  the  reaction  may  proceed:  (1)  the  a  receptors  exclusively 
may  be  present;  (2)  besides  these,  the  organism  may  contain  the 
same  group  a  which  is  present  in  the  injected  blood-cells. 

We  shall  study  these  two  cases  separately  and  begin  with  the 
simpler,  in  which  only  a  receptors  are  present.  In  this  case  the 
conditions  for  the  formation  of  a  hsemolysin  are  given  and  the  bind- 
ing, hyper-regeneration,  and  final  thrusting-off  of  the  a  receptors 
will  follow.  This  newly  formed  immune  body,  in  conjunction  with 
the  complement  always  normally  present,  will  dissolve  all  those  goat 
blood-cells,  and  only  those,  which  possess  the  group  a.  But  as 
this  group  oc,  according  to  our  assumption,  is  completely  absent  in 
the  organism  of  the  animal  itself,  the  immune  body  fails  here  to 
find  any  point  of  attack.  The  immune  body  therefore  will  accu- 
mulate in  the  blood  without  hindrance  and  without  causing  the 
slightest  damage  to  the  organism.  This  case  is  the  one  which  applies 
to  the  examples  of  isolysin  formation  described  by  us,  for  it  is  the 


32  COLLECTED  STUDIES  IN  IMMUNITY. 

only  one  which  fulfills  the  conditions  necessary  for  a  permanent 
existence  of  a  free  hsemolysin. 

The  course  of  the  reaction,  however,  is  entirely  different  in  the 
second  case,  i.e.,  when  the  group  a  of  the  foreign  blood-cells  which 
fits  into  the  receptor  group  is  found  also  in  the  organism  of  the 
animal  injected,  being  present  in  its  blood-cells  and  tissues.  In  this 
case,  groups  fitting  to  one  another  would  be  present  in  the  same 
organism.  A  pregnant  example  is  seen  in  this,  that  both  the  rennin 
and  the  antirennin  group  may  occur  simultaneously  in  the  organism. 
In  fact  we  believe  that  this  simultaneous  occurrence  of  such  corre- 
sponding groups  is  a  very  frequent  phenomenon  in  the  economy  of  the 
organism,  and  that  it  occurs  especially  in  those  cases  in  which  a 
certain  cell  is  dependent  for  its  nutrition  on  the  products  of  a  dif- 
ferent kind  of  cell.1 

If  this  is  the  case,  i.e.,  when  group  a.  is  present  in  the  organism 
beside  the  receptor  group,  the  first  phase  will  proceed  just  as  in  the 
first  case.  There  will  be  a  binding,  regeneration,  and  thrusting-off 
of  the  receptor  as  immune  body.  The  difference  in  the  course  of 
the  reactions  becomes  manifest  in  a  second  phase  in  which  these 
thrust-off-  receptors  are  taken  up  by  group  a. 

Under  certain  circumstances  this  might  lead  to  serious  injury, 
namely,  when  the  thrusting-off  of  the  receptors  as  immune  bodies 
occurs  so  suddenly  that  the  organism  is  overwhelmed,  the  red  blood- 
cells  anchoring  the  receptor  group  and  being  dissolved  by  the  ever- 
present  complement.  In  this  case,  then,  an  autolysin  could  develop. 
But  this  result  need  not  of  necessity  ensue.  It  can  be  prevented, 
for  example,  if  at  first  only  small  amounts  of  the  liberated  receptor 


1  In  contrast  to  this  we  shall  have  to  assume  that  singular  haptophore  groups 
occur  wherever  it  is  designed  to  catch  hold  of  certain  exogenous  constituents 
of  the  nourishment.  In  immunization  it  is  of  some  consequence  whether  a 
singular  group  functionates  as  receptor,  or  one  which  corresponds  to  another. 
The  former  is  probably  the  case  with  the  toxin,  and  this  permits  of  an  extraor- 
dinary increase  in  the  production  of  antitoxin,  being  limited  by  no  regulating 
contrivance.  If,  however,  the  antigroup  is  present  in  the  organism,  owing  to 
secondary  influences,  a  regulatory  production  of  new  antigroups  will  occur. 
This  might  be  the  reason  why  it  is  apparently  impossible  to  increase  the  pro- 
duction of  antirennin  to  any  desired  degree.  The  antirennin  finds  the  corre- 
sponding rennin  group  in  the  organism  and  causes  the  production  and  thrusting 
off  of  this  group.  As  a  result  of  this  series  of  changes  we  find  at  one  time  that 
the  serum  of  an  animal  contains  free  antirennin,  at  another  time  that  rennin 
is  being  excreted  by  the  urine. 


STUDIES  OX  HAEMOLYSIS.  33 

(immune  body)  reach  the  tissues.  This  would  effect  a  production 
and  thrusting-off  of  the  corresponding  group  a,  which  would  then 
circulate  as  an  antiautolysin  and  serve  to  switch  the  autolysin  there- 
after formed,  away  from  the  blood-cells.  Be  this  as  it  may,  whether 
the  organism  be  injured  as  a  result  of  an  acute  flooding  with  the 
liberated  receptors,  or  whether  this  injury  be  prevented  by  the  slow 
course  of  the  reaction,  the  end  result  in  the  second  case  will  regularly 
be  a  development  of  an  antiautolysin.1 

The  three  possibilities,  therefore,  which  present  themselves  on 
the  injection  of  blood  of  the  same  species  are:  1,  the  failure  of  any 
formation  of  hcemolysin;  2,  the  formation  of  an  isolysin;  3,  the  develop- 
ment of  an  antiautolysin. 

Each  haptophore  group  of  the  red  blood-cells  (and  we  have  reason 
to  assume  a  large  number  of  different  groups  in  each  erythrocyte 
of  every  species)  will  have  to  react,  in  the  animal  body,  according 
to  the  above  scheme.  This  leads  to  a  large  number  of  possibilities. 
If,  for  example,  an  injected  blood-cell  possesses  three  haptophore 
groups,  a,  /?,  7-,  it  will  be  possible  for  a  to  cause  the  development  of 
an  isolysin,  /?  an  antiautolysin,  while  ?  produces  no  effect  whatever. 

This,  of  course,  complicates  the  problem  extraordinarily.  A 
multiplicity  of  variations  is  presented  whose  complete  investigation 
would  require  a  great  deal  of  time  and  labor.  The  three  cases  above- 
mentioned,  however,  amply  suffice  to  explain  all  our  observations 
thus  far.  The  differences  in  the  three  isolysins  previously  described 
are  to  be  ascribed  to  the  action  of  three  different  haptophore  groups 
of  the  blood-cells;  and  the  fact  that  the  same  blood  injected  into  two 
animals  causes  the  development  of  different  isolysins  is  to  be  explained 
by  the  individual  differences  in  the  receptors.  Finally,  the  failure 
of  any  isolysin  reaction  whatever  would  correspond  to  an  absence 
of  suitable  receptors. 

1  The  cases  here  discussed  are  of  general  significance  for  the  question  whether 
hsemolysins  exist  at  all,  and  they  determine  also  the  conditions  under  which 
the  hspmolysins  of  normal  serum  are  capable  of  existence  (see  also  the  second 
communication,  pages  11-23).  The  fact  that  a  normal  haemolysin  dissolves  the 
blood-cells  of  foreign  species  but  spares  its  own  blood-cells,  that,  for  example, 
dog  serum  dissolves  guinea-pig  blood,  rat  blood,  goat  blood,  sheep  blood,  etc., 
but  not  dog  blood,  is  only  a  single  instance  of  the  above-mentioned  general 
law  that  autolysins  are  not  capable  of  existence  in  an  organism;  for  the  presence 
of  receptors,  which  is  essential  to  the  production  of  autolysins,  would,  if  the 
autolysins  should  develop,  soon  result  in  a  compensation  by  means  of  anti- 
autolvsin  formation. 


34  COLLECTED  STUDIES  IN  IMMUNITY. 

Though  the  existence  of  the  antiautolysin  is  theoretically  pos- 
sible, we  have  thus  far  been  unable  to  demonstrate  it.  To  do  this 
it  would  first  be  necessary  to  get  hold  of  an  appropriate  autolysin. 
The  possibility  of  getting  this,  however,  is  only  conceivable  in  such 
favorable  cases  where  the  autolysin  might  be  produced  critically 
and  in  large  amounts.  This  certainly  did  not  occur  in  the  cases 
observed  by  us,  and  we  were  therefore  compelled  to  try  a  different 
method  to  demonstrate  such  an  antibody.  We  know  of  a  number 
of  haemolysins  which  dissolve  goat-blood  and  which  therefore  fit 
to  certain  haptophore  groups  of  the  goat  blood  cells.  It  is  con- 
ceivable that  one  of  these  haptophore  groups  is  identical  with  that 
of  the  autolysin  sought  for,  and  that  an  antiautolysin  fits  this 
group.1 

With  this  end  in  view  we  have  made  a  number  of  experiments 
and  tested  the  action  of  our  inactive  goat  serum  on  the  goat-blood- 
dissolving  action  of  dog  serum,  pig  serum,  and  goose  serum  ard  on 
the  serum  of  a  rabbit  treated  with  goat  blood.  The  results,  however, 
were  not  positive.  From  this,  of  course,  we  are  not  to  conclude 
that  antiautolysins  are  not  at  all  present  in  these  cases.  We  shall 
rather  extend  and  vary  our  experiments  in  all  possible  directions 
until  a  lucky  coincidence  leads  us  to  find  a  fitting  hsemolysin. 

Perhaps  the  most  important  of  the  questions  thus  presented  is 
whether  this  deficiency  of  binding  groups  in  the  red  cells  is  performed, 
or  whether  it  is  due  to  a  new  regulating  power  of  the  organism.  In 
the  latter  case  this  power  would  be  suited  in  the  highest  degree  to 
protect  the  body  even  without  the  formation  of  an  antiautolysin. 

In  one  case,  to  be  sure  (goat  E),  it  seemed  as  though  the  insen- 
sitiveness  was  developed  only  in  response  to  the  blood  injection. 
The  blood-cells  of  this  goat  (the  goat  had  been  repeatedly  injected) 
were  primarily  sensitive  to  isolysins  A  and  B.  After  the  injection 
there  developed  a  complete  insensitiveness  to  isolysin  B,  although 
the  sensitiveness  to  A  remained.  In  this  case  an  isolysin  was  not 
developed,  so  that  if  accidental  circumstances  are  excluded,  it  appears 
as  if  under  the  influence  of  this  blood  injection  a  direct  change  or 
destruction  of  the  binding  groups  had  taken  place. 

We  may  perhaps  also  assume  that  the  complete  insensitiveness 

1  The  multiplicity  of  the  combining  groups  of  the  blood-cells  is  well  illustrated 
by  the  blood  of  buck  A.  This  blood  is  insensitive  to  the  isolysins  mentioned. 
Independently  of  this,  however,  it  retains  complete  sensitiveness  to  hsemolysins 
of  a  different  origin,  pig  serum,  goose  serum,  specific  goose  serum  from  rabbits. 


STUDIES  OX  HXMOLYSIS.  35 

of  buck  A  to  isolysin  B  is  a  secondary  one,  due  to  the  treatment; 
for  thus  far,  among  the  many  normal  goats  examined,  we  have  failed 
to  find  a  single  one  whose  blood-cells  are  completely  insensitive  to 
isolysins  A  or  B. 

These  phenomena  require  further  and  more  extended  investi- 
gation, and  hi  this  we  are  at  present  engaged. 

In  closing  we  should  like  to  point  out  that  the  difference  between 
isolysins  and  autolysins  emphasized  by  us  makes  several  recent 
attempts  directed  to  the  solution  of  certain  pathological  processes, 
particularly  those  of  autointoxication  hi  man,  appear  questionable. 
It  has  frequently  been  ascertained  that  serum  secretions  and  excre- 
tions of  the  diseased  body  are  poisonous  hi  animal  experiments, 
and  the  conclusion  has  been  drawn  that  the  substances  to  which 
this  poisonous  action  is  due  must  exert  an  injurious  effect  on  the 
organism  of  the  patient.  From  the  above  analysis  we  see  that  this 
conclusion  is  not  at  all  imperative.  If,  for  example,  the  serum  of 
a  scarlet  fever  patient  is  especially  toxic  to  guinea-pigs,  it  is  possible 
that  the  same  may  be  absolutely  harmless  to  the  patient  himself. 
Even  if  one  demonstrates  that  the  serum  of  anaemic  individuals  dis- 
solves the  blood-cells  of  other  individuals,  it  does  not  prove  that 
this  property  is  of  any  significance  for  the  origin  of  the  anaemia. 
On  the  contrary  it  is  highly  probable  that  this  haemolysin  is  only 
an  isolysin  and  not  an  autolysin. 

The  above  experiments  may  suffice  to  show  how  very  complicated 
the  conditions  are  when  the  material  of  its  own  body  is  absorbed 
by  an  organism.  Drawing  a  general  conclusion,  however,  we  may 
say  that  such  an  absorption,  which  as  already  stated  extends  to 
the  greatest  variety  of  cells  and  occurs  hi  numerous  instances,  will 
not  as  a  rule  lead  to  permanent  injury  of  the  organism,  owing  to 
the  formation  of  reaction  products.  Only  when  the  internal  regu- 
lating contrivances  are  no  longer  intact  can  great  dangers  arise. 
In  the  explanation  of  many  disease  phenomena  it  will  in  the  future 
be  necessary  to  consider  the  possible  failure  of  the  internal  regu- 
lations as  well  as  the  action  of  directly  injurious  exogenous  or  endog- 
enous substances. 


IV.  CONTRIBUTIONS   TO  THE    STUDY  OF  IMMUNITY.* 

By  Dr.  von  DUNGERN,  University  of  Freiburg,  Germany. 

A.    New  Experiments  on  the  Side-chain  Theory. 

THE  combining  experiments  of  Ehrlich  and  Morgenroth2  showed 
conclusively  that  the  two  components  of  an  immune  serum  necessary 
for  haemolysis  and  first  demonstrated  by  Bordet,  namely  the  immune 
body  which  withstands  heating  to  56°  C.  and  the  complement  (addi- 
ment)  which  is  present  even  in  normal  serum,  can  under  certain  cir- 
cumstances exist  in  a  serum  side  by  side,  uncombined.  The  immune 
body  possessed  a  strong  affinity  to  the  blood-cells  to  which  it  spe- 
cifically belonged,  being  anchored  by  these  cells  at  0°  C.  and  thus 
separated  from  the  complement,  which  latter  remained  in  the  serum. 
The  complement  was  abstracted  from  the  serum  by  the  erythrocytes 
only  at  higher  temperatures  provided  the  immune  body  was  present 
at  the  same  time.  When  the  latter  was  absent  the  blood-cells  failed 
to  combine  with  any  complement  whatever.  The  complement, 
therefore,  because  of  its  lack  of  affinity,  was  unable  to  act  on  the 
blood-cells,  and  likewise  the  mere  anchoring  of  the  immune  body 
by  the  blood-cells,  without  the  presence  of  the  complement,  was 
unable  to  effect  any  haemolysis.  The  most  plausible  explanation 
for  these  facts  was  this,  that  solution  is  effected  by  the  complement, 
but  that  this  substance  first  requires  the  immune  body  to  enable  it 
to  lay  hold  of  the  blood-cells. 

Bordet3  has  assumed  that  the  immune  body,  independently  of 
the  complement,  combines  with  the  substance  of  the  erythrocyte 
and  so  changes  this  that  it  (the  erythrocyte)  now  combines  with 
the  complement.  Against  this  assumption  must  be  urged  that 
as  a  matter  of  fact  there  is  a  definite  relation  between  immune  body 

1  Reprinted  from  the  Munchener  med.  Wochensohrift,  No.  20,  1900. 

2  See  pages  1-23  of  this  volume. 

3  Annales  de  1'Institut  Pasteur,  1899,  No.  14. 

36 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  37 

and  complement  of  the  same  species.  An  immune  serum  inactivated 
by  heating  to  56°  C.  can  always  be  reactivated  by  the  addition  of 
fresh  blood  serum  from  an  animal  belonging  to  the  same  species  as 
that  from  which  the  immune  serum  was  derived.  The  complements 
of  other  species  of  animal,  however,  reactivate  this  immune  body 
in  the  most  divergent  manner. 

The  results  of  the  combining  experiments  were  readily  harmonized 
with  the  requirements  of  the  side-chain  theory.  The  immune  body 
is  nothing  but  a  side-chain  with  two  haptophore  groups,  which  has 
been  produced  in  excess  and  thrust  off  into  the  blood.  One  of  these 
haptophore  groups  possesses  a  strong  chemical  affinity  for  the  corre- 
sponding group  of  the  erythrocyte,  and  ordinarily  it  serves  to  anchor 
nutritive  material  possessing  corresponding  haptophore  groups  to 
the  cells.  The  other  haptophore  group  is  able  to  combine  more 
or  less  completely  with  complement  present  in  the  serum.  It  is 
probably  designed  to  collect  from  the  blood  plasma  the  ferment- 
like  complement,  which,  by  splitting  up  the  nutritive  substances, 
makes  their  assimilation  possible. 

There  is,  however,  another  view  to  take  of  these  phenomena.  It 
is  comprehensible  that  the  cell,  as  such,  produces  the  two  compo- 
nents necessary  for  haemolysis  simultaneously  and  in  relation  with 
each  other,  in  such  fashion  that  in  the  assimilation  of  the  substances 
anchored,  it  constantly  produces  the  complement  required  by  means 
of  its  own  activity  and  does  not  depend  on  the  supply  from  with- 
out, from  the  blood  plasma.  The  assumption  of  such  a  complex 
system — in  which  two  members  so  intimately  connected  are  yet 
so  readily  dissociated — offers  difficulties  which  it  is  unnecessary  to 
discuss  further,  especially  because,  as  will  be  seen  later,  experi- 
ments have  precluded  this  possibility. 

If,  however,  the  side-chain  theory  is  correct  we  shall  expect: 

1.  That  immune  body  and  complement  are  not  present  in  the 
immune  serum  in  equivalent  proportions,  but  that  quantitatively 
they  may  be  independent  of  each  other. 

2.  That  the  same  group  of  the  red  blood-cells  which  in  haemolysis 
combines  with  the  immune  body  causes  the  production  of  the  im- 
mune body. 

3.  That  cells  which  possess  such  form  of  complex  side-chains  are 
enabled  by  the  presence  of  the  complementophile  groups  to  abstract 
complement  from  the  blood  serum. 

1.  The  question  whether  hi  the  immunity  reaction  only  the  inao 


38  COLLECTED  STUDIES  IN  IMMUNITY. 

tive  immune  body  is  produced,  which  then  combines  secondarily 
with  the  complement  present  in  the  blood,  or  whether  the  two  sub- 
stances reach  the  circulation  together,  can  under  favorable  con- 
ditions  be  answered  by  an  exact  quantitative  analysis  of  the  immune 
serum  for  immune  body  and  complement. 

'  I  have  therefore  treated  a  number  of  rabbits  with  cattle  blood, 
cow's  milk,  and  tracheal  epithelium  of  cattle,  and  examined  the 
hsemolytic  immune  sera  thus  obtained  for  their  exact  content  in  im- 
mune body  and  complement.  Corresponding  to  the  material  injected, 
the  erythrocytes  of  cattle  were  always  used  as  a  reagent.  The 
method  employed  was  the  same  in  all  cases ;  decreasing  amounts  of  the 
various  blood  sera  were  mixed,  each  with  one-half  cc.  5%  cattle 
blood  dilution  (in  0.8%  NaCl  solution),  the  mixture  was  kept  at 
37°  C.  for  two  hours  and  tested  for  haemolysis.  It  was  then  very 
readily  proven  that  an  equivalence  between  immune  body  and  com- 
plement does  not  at  all  exist. 

If  such  an  equivalence  were  present,  the  immune  body  of  the 
fresh  immune  serum  would  be  completely  saturated  with  complement 
and  would  not  become  more  active  by  the  further  addition  of  com- 
plement. The  experiments  demonstrated  the  contrary,  for  in  some  cases 
the  power  of  the  immune  sera  was  markedly  incraseed  by  the  addi- 
tion of  normal  rabbit  serum,  which,  hi  the  doses  employed,  was  not 
itself  able  to  effect  the  slightest  solution  of  the  cattle  blood-cells. 
For  example,  if  the  fresh  serum  of  a  rabbit  which  had  been  treated 
with  cattle  blood  was  able  to  make  ten  times  its  volume  of  a  5% 
cattle  blood  mixture  completely  laky,  the  same  serum  on  the.  addi- 
tion of  a  sufficient  amount  of  complement  was  able  to  dissolve  320 
times  its  volume.  On  comparing  the  various  immune  sera  with  each 
other,  it  is  seen  that  this  increase  in  the  hsemolytic  action  on  the 
addition  of  complement  is  in  direct  proportion  to  the  amount  of  im- 
mune body  present. 

The  experiments  therefore  prove  that  quantitatively  the  immune 
body  is  entirely  independent  of  the  complement. 

We  can,  however,  go  further  and  determine  quantitatively  the 
exact  amount  of  complement  contained  in  the  normal  serum  on  the 
one  hand  and  in  the  immune  serum  on  the  other. 

The  amount  of  complement  contained  in  the  various  normal 
sera  was  determined  by  always  testing  with  the  same  amount  of  a 
blood  immune  body.  In  fixing  such  a  standard  serum  it  is  only  neces- 
sary to  take  as  a  measure  the  action  of  an  immune  body  saturated  with 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  39 

complement,  for  equal  amounts  of  immune  body  act  differently  with 
different  amounts  of  complement.  In  all  my  tests  on  the  amount  of 
complement  contained  in  a  serum,  I  used  so  much  inactivated  blood 
immune  serum  that  the  immune  body,  when  saturated  with  com- 
plement, could  dissolve  sixteen  times  the  amount  of  blood  present. 

The  experiments  demonstrated  that  the  amount  of  complement 
contained  in  normal  rabbit  serum  is  fairly  constant,  and  even  in 
different  animals  is  not  subject  to  great  fluctuations.  Proceeding 
as  just  described,  it  was  found  that  complete  solution  took  place 
in  all  cases  on  the  addition  of  1/4o  to  1/2o  cc.  normal  serum.  Within 
definite  limits  therefore  the  complement  hi  rabbit  blood  seems  fixed. 

The  amount  of  complement  contained  in  immune  serum  could  be 
determined  by  comparing  the  haemolytic  action  of  the  fresh  serum 
with  its  action,  after  inactivation  (by  heating  for  twenty  minutes 
to  56°  C.).  on  the  addition  of  various  amounts  of  normal  rabbit  serum, 
the  complement  content  of  which  was  known. 

The  serum  of  the  rabbits  treated  with  cattle  blood,  serum  which  had 
been  shown  to  contain  such  a  large  excess  of  immune  body,  was  tested 
1}  2,  8,  4,  11,  and  14  days  after  the  injection  and  failed  in  all  of  the 
numerous  cases  to  show  even  a  trace  of  increase  in  the  amount  of  com- 
plement it  contained.  A  peculiar  state  of  affairs  is  thus  presented. 
Since  haemolytic  action  is  dependent  on  the  immune  body  so  far 
as  this  can  combine  with  the  complement,  we  see  that  the  haemolytic 
action  of  fresh  immune  serum  can  be  increased  only  up  to  a  certain 
point,  determined  by  the  amount  of  complement  contained  in  the 
normal  blood  serum.  All  additional  amounts  of  immune  body 
formed  hi  the  course  of  the  immunity  reaction  therefore  remain 
latent,  and  manifest  their  action  only  when  the  immune  body  is 
brought  into  combination  with  greater  amounts  of  complement. 
This  can  be  done  artificially,  in  test  tube  experiments,  by  the  addi- 
tion of  normal  serum,  or  experimentally  by  injecting  the  immune 
body  into  a  suitable  animal  body.1 

Immune  serum  therefore  differs  from  normal  serum  only  in  its  con- 
tent of  inactive  immune  body.  Accordingly,  in  the  immunity  reaction, 
only  inactive  immune  body  is  produced  by  the  cells  in  excess.  This 

1  So  also  the  earlier  observations,  as  those  of  R.  Pfeiffer,  on  cholera  serum, 
my  own  on  epithelial  immune  serum,  and  those  of  Moxter  on  antispermatozoa 
serum,  in  which  the  immune  sera,  in  themselves  little  or  not  at  all  active,  showed 
their  full  power  when  injected  into  fitting  animal  bodies,  are  to  be  explained 
by  the  relative  poverty  of  these  sera  in  preformed  complement. 


40  COLLECTED  STUDIES  IN  IMMUNITY. 

result  is  easily  understood  on  the  basis  of  the  side-chain  theory, 
if  we  assume  that  the  production  of  the  complement  is  entirely  inde- 
pendent of  the  binding  of  the  injected  substances  by  the  side-chains, 
and  is  probably  referable  to  other  cells.  If  the  production  and 
thrusting  off  of  the  particular  side-chains  exceeds  a  certain  limit,  these 
side-chains  will  fail  to  find  in  the  blood  serum  any  more  complement 
whose  haptophore  group  is  still  available.  The  disproportion  between 
immune  body  and  complement  then  sets  in.  This  will  be  most 
marked  in  those  cases  in  which  the  normal  serum  contains  but  little 
complement  and  in  which  a  considerable  production  of  immune  body 
can  be  effected. 

2.  Certain  experiments  which  I  have  described  in  a  previous  com- 
munication regarding  globulicidal  action  of  the  animal  organism  1  led 
me  to  the  view  that  the  immune  body  combines  with  a  particular 
group  of  the  blood-cells  and  thus  leads  to  their  solution.  This  con- 
ception was  based  on  the  fact  that  a  specific  affinity  exists  between 
erythrocyte  and  the  corresponding  immune  body,  which  affinity  must 
be  the  same  in  the  production  as  in  the  action  of  the  immune  body. 
According  to  the  side-chain  theory  just  this  affinity  is  the  driving 
force  which  on  the  one  hand  anchors  the  corresponding  group  of 
the  erythrocyte  to  the  preformed  side-chains  (such  side-chains  when 
thrust  off  constituting  the  immune  body),  and  on  the  other,  in 
haemolysis,  anchors  the  immune  body,  and  with  it  the  complement, 
to  the  blood-cells. 

It  must  always  be  conceded  to  the  opponents  of  this  view  that  the 
evidence  to  prove  such  complicated  processes  as  will  develop  in  the 
cells  after  inoculations  of  blood  into  an  animal  body  will  not,  perhaps, 
be  absolutely  conclusive.  If  one  were  willing  to  forego  an  explana- 
tion of  the  specificity,  one  could  assume  that  the  immunity  reaction 
is  based  on  an  increase  of  the  normal  function  of  certain  cells  whose 
products  are  formed  without  requiring  a  certain  group  to  fit  into  a 
corresponding  one. 

It  was  therefore  of  great  interest  to  be  able  to  show  experimentally 
that  the  group  which  in  haemolysis  combines  with  the  immune  body 
actually  gives  rise  to  the  production  of  the  immune  body.  This 
demonstration  was  effected  by  injecting  blood  together  with  inac- 
tivated blood  immune  serum. 

If  the  development  of  the  antibody  is  independent  of  the  group 

4  Munch,  med.  Wochenschrift,  1899,  Nos.  13  and  14. 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  41 

to  which  the  immune  body  is  attached,  the  immunity  reaction 
will  be  exactly  the  same  whether  the  injected  blood  is  loaded  with 
immune  body  or  not.  If,  however,  the  production  of  the  immune  body 
is  dependent  entirely  on  the  molecular  group  for  which  the  immune 
body  possesses  a  specific  affinity,  no  immune  body  will  be  developed 
when  a  sufficient  amount  of  inactivated  blood  immune  serum  is 
added  to  the  injected  blood,  since  the  group  is  already  occupied  by 
immune  body  and  no  longer  offers  the  cells  a  point  of  attachment. 

The  experiments  completely  confirm  the  latter  assumption. 
When  the  blood  loaded  with  immune  body  was  injected,  no  immune? 
body  whatever  was  developed  in  the  injected  animal;  whereas  in  a  con- 
trol rabbit,  injected  with  exactly  the  same  amount  of  cattle  blood 
(30  cc.),  but  without  immune  body,  so  much  was  produced  that 
the  serum  eleven  days  after  the  injection  was  able  to  dissolve  com- 
pletely eight  times  its  volume  of  full  blood  provided  sufficient  com- 
plement was  added. 

This  fact,  like  many  others,  speaks  against  the  idea  that  the 
immune  bodies  or  the  analogous  antitoxins  are  not  reaction  products 
of  the  organism  but  are  derived  by  modification  from  the  substances 
introduced,  a  view  still  maintained  by  certain  high  authorities.  The 
phenomenon,  however,  is  readily  explained  on  the  basis  of  the  side- 
chain  theory.  Since  the  particular  groups  of  the  erythrocytes, 
which  otherwise  give  rise  to  the  immunity  reaction,  are  already 
occupied  by  immune  body,  it  is  impossible  for  them  to  be  bound 
by  the  side-chains,  which  are  absolutely  similar  to  the  immune  body. 

3.  According  to  the  researches  of  Ehrlich  and  Morgenroth,  the 
erythrocytes  of  sheep  possess  no  affinity  whatever  for  the  complement 
of  normal  goat  serum.  If  instead  of  sheep  blood-cells,  one  employs 
those  of  cattle  and  allows  them  to  act  on  rabbit  blood  serum,  exactly 
the  same  thing  will  be  observed;  the  rabbit  blood  serum,  centrifuged 
after  prolonged  contact  with  the  blood-cells,  shows  no  diminution 
in  the  content  of  complement.  //,  however,  other  cells,  e.g.,  ciliated 
epithelium  from  the  trachea  of  cattle,  be  mixed  with  rabbit  serum,  the 
result  is  directly  opposite,  the  complement  decreasing,  and  even  under 
some  circumstances  disappearing  entirely.  In  like  manner  the  rabbit 
serum  may  lose  its  complement  through  the  action  of  other  cells. 
In  the  case  of  various  mammals  and  birds,  every  one  of  the  organs 
tested — liver,  spleen,  kidney,  testis,  lung,  and  brain — was  able  to 
abstract  more  or  less  complement  from  the  rabbit  serum.  Yeast 
cells  and  fission-fungi  were  also  able  to  effect  this.  Especially  remark- 


42  COLLECTED  STUDIES  IN  IMMUNITY. 

able,  however,  is  the  fact  that  the  body  cells  of  the  same  animal 
are  able  to  produce  this  phenomenon. 

Exact  quantitative  examinations  showed  that  there  were  dis- 
tinct differences.  The  spleen  and  kidney  of  a  rat,  for  example,  were 
more  strongly  active  than  the  same  organs  of  a  guinea-pig,  while 
the  liver  tissue  of  the  two  species  possessed  equal  activity;  the 
spleen  and  kidney  of  the  rat  abstracted  more  complement  from 
rabbit  serum  than  did  the  same  quantity  of  liver  tissue,  whereas 
in  the  guinea-pig  the  liver  acted  more  strongly  than  the  spleen,  and 
the  latter,  again,  more  strongly  than  the  kidney.  Virulent  cholera 
vibrios  acted  only  one-quarter  as  strongly  as  the  completely  avirulent 
•"  cholera  Calcutta."  (The  number  of  active  individuals  could  not, 
of  course,  be  regarded.)  Yeast  cells  were  weakly  active,  anthrax 
bacilli  strongly  so.  In  the  case  of  anthrax  bacilli  I  tested  the  action 
of  heat  on  this  property  to  abstract  complement  from  rabbit  serum, 
and  found  that  it  is  not  destroyed  by  heating  the  bacilli  for  twenty 
minutes  to  56°  C.,  but  that  it  is  destroyed  by  heating  them  for  only 
a  short  time  to  98°  C.  But  the  property  of  the  cells  to  abstract 
complement  from  rabbit  serum  is  lost  not  only  through  the  action 
of  heat,  but  also  when  the  particular  cells  previous  to  their  mixture 
with  rabbit  serum  have  been  allowed  to  remain  in  contact  with  another 
serum.  For  example,  1  grm.  finely  crushed  kidney  tissue  of  cattle 
is  mixed  with  2  cc.  cattle  serum,  allowed  to  act  at  37°  C.  for  half  an 
hour  and  then  separated  from  the  serum  by  centrifuge.  If  2  cc. 
rabbit  serum  are  now  added  to  the  sediment,  and  this  is  allowed 
to  stand  for  half  an  hour  at  37°,  it  will  be  found  on  testing  with  cattle 
blood  immune  body  that  there  is  no  diminution  of  complement 
content;  but  such  a  diminution  does  occur  when,  with  exactly  the 
same  procedure,  8  p.  m.  NaCl  solution  is  used  in  place  of  the  cattle 
serum. 

These  phenomena  are  best  explained  by  assuming  that  the  cells 
in  question,  in  contrast  to  the  erythrocytes,  possess  groups  which 
have  a  very  close  chemical  relation  to  those  of  the  complement  which 
reactivates  the  cattle  blood  immune  body.  The  affinity  of  the  cells 
may,  in  fact,  be  greater  for  the  complement  than  for  any  immune 
body  directed  against  other  cells  of  the  same  animal  species.  For 
example,  if  we  add  ciliated  epithelial  cells  from  the  trachea  of  cattle 
to  an  immune  serum  derived  from  a  rabbit  by  treatment  with  cattle 
blood,  we  shall  under  favorable  circumstances  find  that  the  immune 
body  has  been  partially,  but  the  complement  completely,  abstracted 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY  43 

from  the  serum.  In  this,  therefore,  the  combining  relations  are 
just  the  opposite  of  those  found  by  Ehrlich  and  Morgenroth  to  exist 
between  blood-cells  and  their  corresponding  immune  body.  The 
tracheal  epithelial  cells  must  therefore  possess  complementophile 
groups.  The  immune  bodies,  which  according  to  the  side-chain  theory 
are  only  the  side-chains  thrust  off  into  the  circulation,  are  similarly 
supplied  with  complementophile  groups.  These  facts  speak  for 
the  correctness  of  the  views  of  Ehrlich  and  Morgenroth,  especially 
when  we  consider  that  a  cell,  corresponding  to  its  many-sided  func- 
tions, possesses  not  merely  one  kind  of  side-chain,  but  side-chains 
of  the  most  highly  developed  form. 

Mammalian  erythrocytes  in  contrast  to  the  tissue  cells  seem 
not  to  possess  complex  side-chains;  and  this  is  readily  understood 
when  we  consider  that  the  red  blood-cells  of  these  animals,  being 
without  a  nucleus  and  unable  to  maintain  their  nutrition  independently 
are  not  complete  analogues  of  the  tissue  cells;  and  further  that 
their  conditions  of  nutrition,  corresponding  to  their  simpler  func- 
tions, must  be  less  complicated  than  those  of  the  typical  tissue  cell. 
Among  the  living  constituents  of  the  body,  the  red  blood-cells  con- 
stitute the  simplest  case  and  are  therefore  particularly  adapted  to 
the  solution  of  many  special  problems  in  immunity,  as  can  be  seen 
from  the  course  of  the  last  experiments. 

The  phenomenon,  that  body  cells  are  able  to  abstract  complement 
from  the  serum,  furnishes  us  with  a  good  explanation  of  the  fact 
that  immune  sera  are  often  so  little  active  in  an  organism  of  a  dif- 
ferent species.  The  immune  body,  which  in  stronger  concentrations  is 
not  saturated  with  complement,  even  when  the  immune  serum  is 
perfectly  fresh,  can  lose  its  complement  entirely  in  the  body  of  an 
animal  of  different  species ;  it  will  therefore  become  active  only  when 
it  finds  a  fitting  complement  in  the  new  organism.  Hence  in  serum 
therapy  it  is  advisable,  as  Ehrlich  has  proposed,  to  employ  for  pur- 
poses of  immunization,  animals  closely  related  to  man,  and  further- 
more to  search  for  anthropostable  complements. 

B.    Phagocytosis  and  Globulicidal  Immunity. 

In  a  previous  communication  *  I  expressed  the  view  that  the  specific 
increase  of  the  globulicidal  function  of  the  organism,  following  the 
introduction  of  chicken  and  pigeon  blood,  is  due  to  the  action  of  the 

1  Miinch.  med.  Wochenschrift,  1899,  Xos.  13  and  14. 


44  COLLECTED  STUDIES  IN   IMMUNITY. 

serum  and  not  to  the  activity  of  the  phagocytes.  That  the  taking 
up  of  the  blood-cells  by  the  phagocytes  in  the  specifically  treated 
guinea-pig  is  necessary  for  the  solution  of  the  blood-cells  was  ex- 
cluded by  the  fact  that  haemolysis  is  also  effected  in  the  peritoneal 
cavity  of  the  animals  apart  from  the  phagocytic  cells.  Furthermore, 
a  transference  by  the  phagocytes  of  the  substances  necessary  for 
solution  was  not  suggested  because  the  exudate,  rich  in  leucocytes, 
which  was  produced  in  specifically  immunized  guinea-pigs  by  in- 
jections of  an  aleuronat  mixture,  showed  a  much  smaller  content 
of  both  immune  body  and  complement  than  the  blood  which  was 
poor  in  leucocytes. 

Metchnikoff  has  objected  to  these  experiments.1  He  states  that 
aleuronat  exudates  contain  principally  microphages,  whereas  the 
blood  is  richer  in  macrophages,  and  that  the  latter  alone  are  con- 
cerned in  haemolysis.  I  have  therefore  tested  the  spleen  (rich  in 
macrophages)  of  normal  rabbits  and  guinea-pigs  with  a  cattle  blood 
immune  body  derived  from  rabbits  in  order  to  determine  the  amount 
of  complement  present.  The  experiments  have  demonstrated  that 
the  spleen  also  contains  much  less  complement  than  the  blood  serum. 
For  example,  1  grm.  finely  crushed  spleen  of  an  exsanguinated  rabbit 
was  mixed  with  4  cc.  of  an  8  p.  m.  NaCl  solution.  This  fluid,  like 
similar  mixtures  derived  from  liver  and  kidney,  when  tested  in  the 
usual  manner  proved  from  eight  to  sixteen  times  weaker  than  the 
blood  serum.  Moreover,  if  the  suspended  organic  particles  were 
first  washed  with  physiological  salt  solution,  they  yielded  no  com- 
plement whatever  to  the  immune  body.  The  spleen  of  a  guinea-pig 
contained  still  less  complement,  although  the  serum  of  this  same 
animal  completely  activated  the  cattle  blood  immune  body  derived 
from  rabbits,  and  did  so  in  even  smaller  quantity  than  the  rabbit 
serum. 

We  must  therefore  in  conformity  with  the  side-chain  theory  look 
to  the  blood  serum  as  the  chief  source  of  complement. 

It  is  self-evident  that  the  complement  cannot  originate  in  the  blood 
plasma;  it  must,  of  course,  be  derived  from  some  kind  of  cells.  How- 
ever, that  it  is  especially  abundant  in  the  phagocytes  is  not  at  all 
borne  out  by  the  above  experiments. 

As  for  the  immune  body,  Metchnikoff  too  believes  this  to  circulate 
free  in  the  blood  plasma.  According  to  his  conception  the  macro- 

1  Annales  de  PInst.  Pasteur,  1899,  No.  10. 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  45 

> 

phages  yield  this  to  the  blood  at  the  end  of  their  intracellular  diges- 
tion. Metchnikoff  bases  this  view  chiefly  on  his  observations  that  the 
destruction  of  avian  blood-cells  in  the  peritoneal  cavity  of  normal 
guinea-pigs  is  effected  exclusively  by  the  macrophages. 

This  statement  is  in  direct  opposition  to  mine,  according  to  which 
even  in  untreated  animals,  the  solution  takes  place  free  in  the  peri- 
toneal exudate  independently  of  the  phagocytes.  I  believe,  however, 
that  these  apparently  contrary  results  can  well  be  harmonized. 

According  to  Metchnikoff  the  solution  of  goose  blood-cells  in 
the  subcutaneous  connective  tissue  of  even  non-immunized  animals, 
is  effected  almost  exclusively  extracellularly.  Haemolysis  in  this 
case  must  be  due  to  a  passage  of  complement  and  interbody  from 
the  blood  into  the  subcutaneous  tissues;  this  will  naturally  proceed 
more  rapidly  when,  as  a  result  of  substances  exciting  inflammation, 
a  stronger  exudation  ensues. 

It  would  be  very  curious  if  the  same  conditions  for  the  passage 
of  haemolytic  substances  from  the  blood  were  not  present  in  the  peri- 
toneal cavity.  We  know,  for  example,  that  Pfeiffer's  phenomenon 
is  especially  marked  in  the  peritoneal  cavity.  As  a  matter  of  fact, 
shortly  after  an  injection  of  avian  blood-cells  into  the  peritoneal 
cavity  of  normal  guinea-pigs,  one  always  observes  free  nuclei,  even 
when  the  serum  has  been  removed  from  the  cells  by  centrifugation. 
Of  this  I  convinced  myseif  by  repeated  observations.  If  one  employs 
blood-cells  of  low  resistance  (chicken-blood),  and  these  in  small 
doses,  they  will  be  degenerated  and  for  the  most  part  dissolved  before 
they  are  taken  up  by  the  macrophages  in  any  considerable  number. 
When  blood-cells  of  greater  resistance  are  employed,  and  these  in 
larger  doses,  the  solution  effected  by  the  body  juices  will  be  com- 
paratively slight  and  occupy  more  time.  The  taking  up  of  these 
cells  by  the  macrophages,  which  Metchnikoff  in  his  splendid  experi- 
ments was  able  to  follow  into  the  organs,  will  then  come  more  to 
the  front. 

If  therefore,  as  a  result  of  experiments  in  which  I  used  sensitive 
blood-cells  in  small  doses,  I  underrated  the  significance  of  phago- 
cytosis, Metchnikoff,  through  the  conditions  in  his  experiments,  fell 
into  the  opposite  error.  The  truth  lies  between  these  views;  in 
the  peritoneal  cavity,  according  to  the  prevailing  conditions,  haemol- 
ysis can  be  effected  free  in  the  peritoneal  exudate  or  in  the  interior 
of  the  macrophage. 

In  any  case,  phagocytosis  is  not  essential  for  the  development  of 


46  COLLECTED  STUDIES  IN  IMMUNITY. 

the  immune  body.  The  immunity  reaction  occurs  even  under  con- 
ditions in  which  phagocytosis  does  not  at  all  enter;  and  if,  accord- 
ing to  the  observations  of  Metchnikoff,  somewhat  less  immune  body 
is  produced  after  subcutaneous  injections  than  after  equal  injections 
peritoneally,  this  may  be  explained  as  follows:  In  consequence  ctf 
the  slower  absorption  from  the  subcutaneous  tissues,  fewer  cells- 
come  into  contact  with  the  group  of  the  erythrocytes  which  excites 
the  immunity  reaction  before  an  excess  of  immune  body  is  thrust 
off  by  these  cells  into  the  blood.  This  immune  body,  of  course, 
prevents  any  further  combination  of  the  group  in  question  with  other 
cells. 

To  what  extent  the  phagocytes  are  concerned  in  the  production 
of  immune  bodies  must  be  determined  separately  in  each  case.  No- 
definite  conclusions  can  be  drawn  from  the  experiments  of  Metchni- 
koff on  guinea-pigs  with  goose  blood-cells,  for  at  no  time  did  the 
organs  of  the  specifically  treated  guinea-pigs  show  a  stronger  glob- 
ulicidal  action  than  those  of  normal  animals,  although  such  an 
increase  in  haemolytic  power  was  exhibited  by  the  blood  serum.  But 
the  observation  has  been  made  that  even  in  normal  animals  the 
organs  rich  in  macrophages  are  able,  in  contrast  to  other  tissues,  to- 
dissolve  goose  blood-cells,  and  this  observation  is  well  adapted 
in  this  case  to  support  the  assumption  of  a  special  significance  of 
the  phagocytes  for  this  function.  However,  that  organs  rich  in 
macrophages  effect  haemolytic  action  is  not  necessarily  the  case.  For 
example,  the  spleen  of  a  guinea-pig  (1  grm.  finely  crushed  spleen 
suspended  in  1  c.c  of  an  8  p.  m.  NaCl  solution),  in  contrast  to  the 
blood  serum  of  the  same  animal  is  not  globulicidal  for  cattle  blood. 
Considering  the  large  number  of  immune  bodies,  it  will  surely 
often  occur  that  the  phagocytes  are  preeminently  concerned  in  the 
production  of  the  immune  body,  especially  since  these  cells  frequently 
come  into  intimate  relations  with  the  injected  substances.  On  the 
other  hand,  it  is  extremely  improbable  that  the  phagocytes  alone 
produce  immune  body.  After  all  that  has  been  said  we  shall  have 
to  bring  this  production  into  relation  with  the  general  conditions 
of  nutrition.  The  most  varied  cells,  according  to  the  kind  of  side- 
chains  they  possess  and  the  affinities  thereby  brought  about,  are 
probably  able  to  produce  immune  body. 

Like  the  closely  related  antitoxic  immunity  reaction,  the  globu- 
licidal and  bactericidal  reactions  rest  on  a  chemical  process  the 
course  of  which  is  best  explained  on  the  basis  of  the  side-chain  theory. 


V.    CONTRIBUTIONS   TO  THE   STUDY  OF    IMMUNITY.1 

By  Dr.  von  DUNGERN,  University  of  Freiburg,  Germany. 

A.    Receptors2  and  the  Formation  of  Antibodies. 

ACCORDING  to  Ehrlich's  view  3  the  antitoxins  are  formed  in  those 
organs  which,  according  to  their  content  of  receptors,  have  bound 
the  toxin.  Roux  and  Borrel  4  in  combating  to  this  view,  have  pointed 
out  that  rabbits  die  of  tetanus  following  an  intracerebral  injection 
of  very  small  doses  of  tetanus  poison,  and  that  therefore  the  brain 
of  these  animals  contains  no  active  antitoxin.  Weigert  5  has  shown 
that  this  phenomenon  entirely  supports  Ehrlich's  theory.  Since 
the  antitoxin  of  the  central  nervous  system,  so  long  as  it  has  not 
been  thrust  off  into  the  blood,  still  functionates  as  receptor,  it  must 
anchor  the  tetanus  poison  to  the  nerve  cells  and  is  therefore  not 
at  all  adapted  to  protect  these  against  the  action  of  the  toxophore 
group.  Furthermore,  the  fact  that  immunized  animals  behave 
similarly  proves  merely  that  in  these  animals,  after  immunization, 
the  ganglion  cells  still  possess  receptors.  According  to  the  side- 
chain  theory  the  antitoxins  present  in  the  blood  act  merely  by  sat- 
isfying the  toxins  which  gain  access  to  the  blood  and  deflect  these 
from  the  organs  still  possessing  receptors  and  hence  still  sensitive. 
The  observations  of  Roux  and  Borrel  are  therefore  in  entire  har- 
mony with  the  views  of  Ehrlich. 

1  Reprint  from  Munch,  med.  Wochenschrift,  No.  28,  1900. 

2  Ehrlich  and  Morgenroth  designate  those  combining  groups  of  the  proto- 
plasmal  molecule  to  which  a  foreign  group,  when  introduced,  attaches  itself 
"RECEPTORS."     See  also  page  24. 

3Klinisches    Jahrbuch,    1897,   Vol.    VI;   Werthbemessung   des   Diphtheric 
Heil-serum,  Jena,  Fischer,  1897. 

4  Annales  de  1'Institut  Pasteur,  1898. 

6  Ergebnisse  der  allgemein.  Pathologic,  etc.     IV.  Jahrgang,  iiber  1897 

47 


48  COLLECTED  STUDIES  IN  IMMUNITY. 

Metchnikoff  1  has  pursued  this  question  as  to  the  origin  of  the 
antitoxins  further.  Since  a  positive  conclusion  did  not  seem  pos- 
sible to  him  by  the  use  of  the  bacterial  poisons,  he  employed  a  specific 
cell  poison,  spermotoxin,  which  can  be  produced  by  treating  guinea- 
pigs  with  the  testicle  and  epididymus  of  a  rabbit.  The  use  of  this 
poison  has  the  advantage  that  the  organs  against  which  it  is  directed 
can  be  removed  from  the  animal  without  serious  injury.  As  the 
injection  of  this  poison  into  the  body  of  male  rabbits  is  followed 
by  the  production  of  an  antibody,  it  was  merely  necessary  to  repeat 
this  procedure  on  castrated  rabbits  to  decide  the  question  whether 
the  antispermotoxin  is  produced  only  by  the  sexual  cells  or  also 
by  other  organs. 

The  results  showed  that  the  sera  of  rabbits  which  had  been  injected 
with  this  spermotoxin  would  protect  rabbit  spermatozoa  against 
the  action  of  the  spermotoxin  no  matter  whether  the  rabbits  from 
whom  these  sera  were  derived  had  been  castrated  or  not. 

According  to  Metchnikoff's  view,  this  is  opposed  to  the  side- 
chain  theory,  "  since,"  as  he  says,  "  an  antitoxin  is  produced  with- 
out the  presence  of  corresponding  receptors  in  the  organism. "  In 
this,  however,  Metchnikoff  starts  with  the  assumption  that  the  spermo- 
toxin is  absolutely  specific  and  that  it  acts  exclusively  on  sperma- 
tozoa. He  believes  that  the  hsemolytic  action  which  he  has  observed 
in  the  spermatozoa  immune  serum  may  be  explained  by  assuming 
that  with  the  injection  of  testis  and  epididymus  red  blood-cells  were 
introduced,  and  that  these  produced  a  haemolysin  entirely  independ- 
ent of  the  spermotoxin.  Further,  he  thinks  that  any  relation  of  the 
spermotoxin  to  other  cells  is  excluded  by  the  fact  that  in  the  serum 
of  guinea-pigs  which  have  been  treated  with  spermatozoa  these 
cells  suffer  no  greater  change  than  they  do  in  normal  guinea-pig 
serum. 

Having  made  observations  in  the  course  of  my  investigations  on 
epithelial  immunization,  which  contradict  these  assumptions  of 
Metchnikoff,  I  feel  compelled  to  explain  my  views  in  order  to  clear 
up  the  entire  matter. 

As  I  have  mentioned  in  a  previous  communication2  the  ciliated 
epithelial  immune  serum  is  able,  besides  its  specific  action  on  ciliated 
epithelium,  to  dissolve  the  red  blood-cells  of  the  same  animal  species. 

1  Annales  de  PInstitut  Pasteur,  1900,  No.  1. 

2  Munch,  med.  Wochenschrift,  1899,  No.  38. 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  49 

This  haemolytic  property  can  in  no  way,  as  Metchnikoff  believes, 
be  due  to  the  introduction  of  erythrocytes  with  the  injection  of  the 
epithelial  cells  into  the  body  of  the  guinea-pig,1  which  introduction 
would  then  lead  to  the  formation  of  a  specific  haemolysin  directed 
against  the  red  blood-cells.  This  possibility  is  at  once  excluded  by 
the  method  of  procedure  in  this  experiment.  For  reasons  of  asepsis, 
the  tracheae  employed  were  scrupulously  cleansed  with  physiological 
salt  solution  and  thus  all  traces  of  blood  adhering  to  the  surface 
were  removed.  The  epithelium  itself  could  not  contain  any  erythro- 
cytes, for  it  was  obtained  by  carefully  scraping  the  surface  layer, 
which  contains  no  blood-vessels.  Errors  due  to  any  admixture  of 
blood,  therefore,  do  not  enter  into  my  experiments.  Besides,  such 
a  strong  haemolytic  action  as  is  manifested  by  the  ciliated  epithelial 
immune  serum  is  never  produced  by  the  injection  of  such  small 
amounts  of  blood.  In  my  experiments  this  action  was  greater  than 
that  following  the  injection  of  2  cc.  of  cattle  blood. 

The  strongest  proof  that  the  blood-dissolving  property  of  the 
ciliated  epithelial  immune  serum  is  independent  of  injected  blood- 
cells  is  afforded  by  the  fact  that  the  haemolytic  immune  body  of 
this  serum  possesses  greater  affinity  for  the  ciliated  epithelium  than 
that  specifically  derived  by  the  injection  of  blood. 

There  is  no  doubt,  therefore,  that  pure  ciliated  epithelial  immune 
serum  possesses  a  haemolytic  action,  and  that,  furthermore,  the 
hsemolysin  produced  by  epithelial  cells  is  different  from  that  pro- 
duced by  blood-cells. 

Moxter  2  made  very  similar  observations  on  spermatozoa  immune 
serum.  He  found  that  the  serum  of  a  guinea-pig  which  had  been 
treated  with  sheep  spermatozoa  dissolves  the  blood-cells  of  sheep; 
and  he  demonstrated  that  the  immune  body  concerned  in  this 
haemolysis  is  completely  bound  by  the  spermatozoa  of  sheep. 

An  absolute  specificity,  so  that,  for  example,  the  immune  body 
produced  by  means  of  ciliated  epithelium  is  bound  only  by  ciliated 
epithelium,  that  produced  by  means  of  spermatozoa  bound  only  by 
spermatozoa,  that  directed  against  red  blood-cells  only  by  erythrocytes, 
without  the  existence  of  any  affinities  between  the  immune  body 
and  other  cells  of  the  same  species,  does  not  therefore  obtain. 

1  Just  as  with  guinea-pigs,  it  is  possible,  by  injecting  rabbits  with  trachea! 
epithelium  of  cattle,  to  produce  a  serum  haemolytic  for  cattle  blood. 
'Deutsche  med.  Wochenschr.,  1900,  No.  1. 


50  COLLECTED  STUDIES   IN  IMMUNITY. 

This,  of  course,  is  readily  understood  by  means  of  the  side-chain 
theory.  One  could  not  well  assume  that  all  the  side-chains  of  a 
certain  group  of  cells  are  entirely  different  from  all  the  side-chains 
of  the  rest  of  the  cells.  It  is  much  more  probable  that  certain  groups 
which  serve  general  functions  of  nutrition  are  common  to  the  majority, 
if  not  to  all,  of  the  cells  of  the  same  animal. 

When,  therefore,  after  the  injection  of  ciliated  epithelial  cells 
we  see  a  hsemolytic  immune  body  develop,  we  may  assume  that 
among  the  groups  of  the  ciliated  epithelial  cell  which  effect  the 
immunity,  there  are  some  which  are  identical  with  those  of  the  red 
blood-cell  or  at  least  closely  related  to  them  chemically. 

If  this  view  is  correct  we  should  expect  that,  conversely,  the 
immune  body  of  an  immune  serum  derived  by  treatment  with  blood, 
would  be  bound  by  ciliated  epithelial  cells  of  the  same  species.  The 
facts  correspond  entirely  with  this  assumption.  According  to  my 
experiments,  epithelial  cells  from  the  trachea  of  cattle  are  able  par- 
tially to  bind  the  blood  immune  body  derived  by  treating  rabbits 
with  cattle  blood.  The  affinity  of  the  ciliated  epithelium  for  the 
blood  immune  body  is,  however,  as  already  mentioned,  less  than 
that  for  the  hsemolytic  ciliated  epithelial  immune  body  of  the  rabbit 
immune  serum. 

With  this  a  further  fact  of  considerable  importance  becomes 
manifest.  Although  the  ciliated  epithelial  cells  are  destroyed  by 
the  ciliated  epithelial  immune  body  (provided  sufficient  complement 
is  present),  it  has  thus  far  been  impossible  to  demonstrate  any  injury 
of  these  cells  resulting  from  the  binding  of  the  active  blood  immune 
body.  The  epithelial  cells  thus  differ  from  the  red  blood-cells,  which 
are  destroyed  even  by  the  antiepithelial  serum.  We  shall  not  enter 
into  an  explanation  of  these  phenomena,  which  point  to  a  multiplicity 
of  antibodies  produced  in  response  to  cell  material.  It  will  suffice 
to  point  out  that  there  is  a  whole  series  of  substances  which  are 
designated  as  blood  poisons,  because  they  attack  especially  the 
red  blood-cells  while  they  have  little  or  no  effect  on  other  cells. 

The  fact  that  the  blood  immune  body  when  supplied  with  com- 
plement is  bound  by  the  ciliated  epithelial  cells  of  cattle  without 
causing  any  apparent  injury,  proves,  at  least,  that  the  phenomenon 
of  toxic  action  in  no  way  shows  whether  or  not  a  toxin  or  toxin-con- 
taining substance  has  been  bound  by  the  cells.  The  appearance  of 
toxic  symptoms,  to  be  sure,  in  the  case  of  antitoxin-forming  poisons, 
is  proof  that  the  poison  has  been  bound.  An  absence  of  toxic  symp- 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  51 

toms  may  not,  however,  at  once  be  ascribed  to  an  absence  of  affinity 
between  cells  and  the  poisonous  substance. 

The  formation  of  an  antibody,  according  to  the  side-chain  theory, 
follows  only  from  the  binding  of  the  haptophore  group  which  excites 
the  immunity,  to  the  corresponding  side-chain,  and  hence  is  not 
directly  dependent  on  the  toxophore  group. 

As  to  which  cells  will  be  able  to  produce  an  antibody  depends, 
therefore,  on  the  possession  of  a  receptor  for  the  haptophore  group 
in  question.  A  highly  toxic  action  of  the  substance  bound  by  the 
cell  is  not  at  all  essential,  and,  is  in  fact,  often  injurious,  as  has  been 
emphasized  especially  by  Knorr.1  This  action,  as  Ehrlich2  has 
shown  hi  his  experiments  on  toxoids,  is  produced  by  a  molecular 
group  entirely  distinct  from  the  haptophore  group  and  having  no 
relation  to  the  antitoxin. 

If  this  law  applies  even  to  the  true  toxins,  we  shall  all  the  more 
have  to  assume  that  it  applies  where  compound  substances,  such  as 
hsemolysin,  epitheliotoxin,  or  spermotoxin  are  concerned.  In  these 
the  toxophore  group  is  only  loosely  combined  with  the  haptophore 
group;  it  is  nothing  but  the  complement,  which,  according  to  my 
researches,3  can  be  bound  by  all  kinds  of  cells,  even  independently 
of  the  immune  body,  and  can,  under  certain  conditions  of  affinity, 
be  separated  from  the  immune  body. 

We  see,  therefore,  that  the  assumption  by  Metchnikoff,  that  the 
spermotoxin  is  related  exclusively  to  the  spermatozoa,  is  incorrect. 
As  against  it  I  have  here  shown  that  a  toxin  obtained  by  immuniza- 
tion with  epithelial  cells  is  able  to  destroy  the  red  blood-cells  in  the 
same  manner  as  a  true  hsemolysin. 

In  the  following  short  communication  I  can  bring  forward  an 
additional  instance  in  Which  the  development  of  a  haemolytic  immune 
body  results  although  the  co-action  of  the  red  blood-cells  is  com- 
pletely excluded.  Even  this  demonstration  proves  that  the  assump- 
tion on  which  Metchinkoff  based  his  objections  to  the  side-chain 
theory  is  contrary  to  the  facts.  The  phenomenon  that  even  in 
castrated  rabbits  an  antispermotoxin  is  formed  is  therefore  readily 
explained  according  to  the  side-chain  theory  by  assuming  that  re- 


1  Munch,  med.  Wochenschr..  1898,  Nos.  11  and  12. 

2  Klin.   Jahrbuch,   1897,  Vol.   VI,  and  Deutsch.   med.   Wochenschr.,   1898, 
No.  38. 

3  See  page  41. 


52  COLLECTED  STUDIES  IN  IMMUNITY. 

ceptors  for  the  immune  body  of  the  spermatozoa  immune  serum 
are  present  not  only  in  the  organs  of  generation  but  also  in  other 
cells  of  the  rabbit.  When,  in  addition,  we  come  to  consider  the 
results  of  these  last  experiments,  we  find  that  the  demonstration 
of  Metchnikoff  (that  even  in  castrated  animals,  in  response  to  treat- 
ment with  spermotoxin,  a  body  is  developed  which  prevents  the 
action  of  the  spermotoxin)  loses  all  value  as  proof  for  the  origin  of 
a  specific  antispermotoxin. 

The  active  spermotoxin  employed  by  Metchnikoff  is  of  course  not 
a  simple  poison;  it  consists,  just  like  a  haemolysin,  of  the  specific 
immune  body  obtained  by  immunization  and  the  complement  present 
in  all  guinea-pig  serum.  Now  it  has  been  shown  independently  by 
Ehrlich  1  and  Bordet  2  that  when  the  complement  is  injected  into 
foreign  species  it  excites  the  production  of  an  anticomplement  which 
inhibits  the  action  of  an  active  immune  body  by  taking  away  the 
complement,  and  that  it  does  this  without  possessing  any  specific 
affinity  to  this  immune  body. 

It  is  therefore  possible  that  the  action  of  the  antispermotoxin 
obtained  by  Metchnikoff  is  to  be  explained  thus:  The  injected 
guinea-pig  serum  by  virtue  of  the  complement  ( Bordet 's  alexin) 
which  it  contains,  causes  the  production  of  an  anticomplement 
serum  which  then  renders  the  complement  of  the  spermotoxin  (de- 
rived from  guinea-pigs)  innocuous.  With  this  idea,  Bordet  has  ex- 
amined an  antihaemolysin,  which  is  analogous  to  the  antispermo- 
toxin, and  has  found  that  the  action  of  the  anticomplement  is  much 
more  pronounced  than  that  of  the  anti-immune  body.  The  forma- 
tion of  an  anticomplement  does  not,  of  course,  according  to  the  side- 
chain  theory,  presuppose  the  presence  of  spermatozoa;  for  accord- 
ing to  my  experiments  the  complement  may  possess  affinities  for  the 
most  varied  cells  of  the  organism. 

Ehrlich's  theory,  that  the  antitoxins  are  produced  by  those 
organs  which  possess  chemical  relations  to  the  toxins,  is  therefore 
in  no  way  affected  by  the  observations  of  Metchnikoff. 

B.    Milk  Immune  Serum. 

After  it  had  been  found  that  it  is  possible  to  produce  a  specific 
immune  serum  by  injecting  guinea-pigs  with  ciliated  epithelium  from 

1  Croonian  lecture,  Royal  Society,  London,  March  1900. 
8  Annal.  de  ITnstitut  Pasteur,  May  1900. 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  53 

the  trachea  of  cattle  it  was  but  a  step  to  employ  epithelial  secretions 
for  the  same  purpose.  In  conjunction  with  this  it  was  of  considerable 
theoretical  interest  to  determine  in  this  very  way  whether  the  specific 
properties  of  cells  are  preserved  in  their  secretion  products. 

I  have  therefore  employed  milk  for  immunization  and  have  first 
treated  guinea-pigs  and  rabbits  with  cow  milk.  The  cow  milk 
immune  serum  thus  obtained  is  able,  so  far  as  I  have  been  able  to 
observe,  to  kill  ciliated  cells  in  the  peritoneal  cavity  of  rabbits,  though 
in  a  smaller  measure  than  the  specific  ciliated  epithelial  immune  serum. 

The  affinities  of  an  immune  serum  are  readily  determined  when 
the  serum,  like  the  ciliated  epithelial  immune  serum  for  example,  acts 
also  on  red  blood-cells,  for  then  this  can  be  used  as  a  reagent.  Cow 
milk  immune  serum  possesses  the  property  to  dissolve  cattle  blood 
in  a  not  inconsiderable  degree.  This  haemolytic  action,  as  in  the 
case  of  the  blood  immune  serum  and  of  the  ciliated  epithelial  immune 
serum,  is  due  not  to  any  increased  content  of  complement  but  to  the 
presence  of  a  specific  immune  body.  Hence  here  also  it  was  pos- 
sible to  compare  the  affinities  of  this  immune  body  (for  the  ciliated 
epithelium  on  the  one  hand  and  for  the  red  bloocj-cells  on  the  other) 
with  the  affinities  of  the  specific  blood  immune  body. 

The  two  immune  sera  obtained  by  injecting  rabbits  with  cow 
milk  and  with  cattle  blood  were  therefore  inactivated,  equal  quan- 
tities of  normal  rabbit  serum  to  serve  as  complement  were  added 
to  them  in  excess,  and  the  mixture  tested  for  its  haemolytic  properties 
on  cattle  blood.  The  cow  milk  immune  serum  usually  showed  such  a 
degree  of  action  that  one  part  of  the  immune  serum  saturated  with 
complement  was  able  to  dissolve  completely  20  parts  of  the  custom- 
ary 5%  cattle  blood  mixture. 

Corresponding  to  this,  therefore,  the  much  stronger  haemolytic 
cattle  blood  immune  serum  was  diluted  with  inactivated  normal 
rabbit  serum  or  with  physiological  salt  solution  until,  with  an  excess 
of  complement,  the  haemolytic  action  of  the  two  sera  on  cattle  blood 
was  exactly  equal. 

When  the  two  immune  bodies  have  in  this  way  been  made  entirely 
equal  so  far  as  the  haemolytic  property  is  concerned,  it  is  possible  to 
exactly  compare  their  chemical  affinities  for  a  particular  group  of 
cells.  It  is  then  easily  demonstrated  that  the  two  haemolytic  immune 
bodies  differ  in  respect  to  their  chemical  relations  to  other  cells  of 
the  same  species. 

Thus  if  equal  quantities  of  ciliated  epithelium  are  added  to  the 


54  COLLECTED   STUDIES  IN  IMMUNITY. 

two  sera  and  the  mixture  centrifuged  some  time'  after,  it  will  be 
found  that  the  milk  immune  body  has  been  completely  abstracted 
from  the  serum,  but  the  blood  immune  body  only  partially  so.  Cili- 
ated epithelium,  therefore,  combines  more  strongly  with  the  milk 
immune  body  than  with  the  blood  immune  body. 

On  the  other  hand,  the  blood  immune  body  possesses  a  greater 
affinity  to  the  erythrocytes  than  does  the  milk  immune  body.  Thus 
if  equal  amounts  of  cattle  blood  are  added  to  the  two  inactivated 
immune  sera  (amounts  which  would  be  completely  dissolved  if  suf- 
ficient complement  were  present),  it  will  be  found  after  a  certain  time 
that  the  blood  immune  body  has  been  completely  bound  by  the 
red  blood-cells,  whereas  the  milk  immune  body  can  still  partially  be 
demonstrated  in  the  serum. 

If  one  tests  a  number  of  different  cow  milk  immune  sera  in  this 
way,  the  results  will  show  marked  variations.  My  experiments  were 
conducted  on  four  different  cow  milk  immune  bodies  which  had 
been  obtained  by  injecting  rabbits  with  cow  milk.  Three  of  these 
showed  considerably  less  affinity  to  the  red  blood-cells  than  did 
the  specific  blood  immune  body  obtained  by  treatment  with  blood. 
The  fourth,  however,  was  bound  by  the  red  blood-cells  in  about  the 
same  degree  as  was  the  blood  immune  body.  On  the  other  hand, 
cases  were  observed  in  which  the  serum  of  rabbits  after  these  had 
been  injected  with  cow  milk  showed  only  a  very  slight  hsemolytic 
action,  and  this  only  on  the  most  sensitive  of  the  blood-cells. 

All  of  these  differences  manifested  themselves  quite  independ- 
ently of  the  cattle  blood  employed  in  the  experiment  and  must  there- 
fore be  ascribed  to  differences  in  the  immune  sera  themselves.  Pos- 
sibly they  are  due  to  variations  in  the  kind  of  receptors,  such  as 
were  found  in  a  marked  degree  in  the  experiments  of  Ehrlich  and 
Morgenroth  on  isolysins.1  The  strong  affinity  of  the  hsemolytic 
milk  immune  body  for  trachea!  epithelium,  however,  was  present 
in  all  the  cases  examined  and  it  did  not  differ  materially  from  the 
chemical  relationship  between  ciliated  epithelium  and  its  specific 
ciliated  epithel  immune  body. 

Hence  by  treatment  with  cow  milk  we  obtain  a  hsemolytic  immune 
serum  which  differs  from  the  blood  immune  serum,  but  cannot  with 
certainty  be  differentiated  from  the  ciliated  epithel  immune  serum. 


1  See  page  23. 


CONTRIBUTIONS  TO  THE  STUDY  OF  IMMUNITY.  55 

The  cow  milk  immune  serum,  owing  to  the  character  of  its  affinities, 
is  to  be  classed  with  the  epithel  immune  serum. 

The  interesting  fact  to  be  deduced  from  this  is  that  milk  con- 
tains the  same  specific  groups  as  the  epithelial  cells  which  produce 
it;  and  this  agrees  very  well  with  histological  observations  accord- 
ing to  which  the  protoplasm  of  the  gland  cells  is  itself  used  in  the 
production  of  the  milk. 

After  having  found  it  possible  to  produce  a  specific  epithel  immune 
serum  by  injections  of  cow  milk,  it  seemed  to  me  that  immunization 
with  human  milk  might  prove  useful  in  the  suppression  of  carcinoma, 
especially  mammary  carcinoma.  Thus  far,  however,  the  treatment 
of  dogs  and  rabbits  with  human  milk  has  not  yielded  an  immune 
serum  haemolytic  for  human  blood,  one  corresponding  to  the  cow 
milk  immune  serum. 


VI.    STUDIES  ON  ILEMOLYSINS.1 

FOURTH  COMMUNICATION. 
By  Professor  Dr.  P.  EHRLICH  and  Dr.  J.  MORGENROTH. 

THE  continued  thorough  study  of  both  natural  hsemolysins  and 
those  produced  by  injections  of  red  blood-cells  leads  to  the  con- 
ception of  an  extraordinary  multiplicity  of  the  substances  which  are 
either  normally  present  in  serum  or  which  we  are  able  at  will  to 
produce  therein.  That  in  the  action  of  the  artificially  developed 
hsemolysins  two  substances  are  always  concerned  may  now  be  regarded 
as  a  fact  supported  by  numerous  individual  observations.  The  two 
substances  are:  (1)  the  specific  immune  body  produced  by  immuniza- 
tion, and  (2)  a  substance,  usually  thermolabile,  contained  even  in  nor- 
mal serum,  our  " complement"  and  the  "alexin"  of  Buchner  and  of 
Bordet.  We  have  shown  that  the  erythrocytes  anchor  the  immune 
body  in  a  specific  manner,  while  they  do  not  combine  with  the  isolated 
complement  as  such.  The  fact  that  the  immune  body  has  been 
bound  by  the  corresponding  erythrocytes  has  been  confirmed  by 
von  Dungern,  Bordet,  and  Buchner.  Out  of  a  fluid  containing  both 
immune  body  and  complement,  at  0°  C.  the  blood-cells  take  up 
only  immune  body,  at  higher  temperatures  both  immune  body  and 
complement.  We  were  able  to  explain  this  phenomenon  only  by 
assuming  that  the  immune  body  possesses  two  haptophore  groups, 
one  of  greater  affinity,  which  is  related  to  a  receptor  of  the  blood- 
cells  and  acts  at  0°  C.,  the  other,  of  less  affinity,  which  combines 
only  at  higher  temperatures  with  a  corresponding  group  of  the  com- 
plement. 

Our  views  can  be  expressed  most  simply  by  means  of  the  fol- 
lowing rough  diagram  (see  figure).  This  will  also  serve  to  show 
the  close  relations  existing  between .  lysins  and  the  true  toxins. 

1  Reprint  from  the  Berlin,  klin.  Wochenschrift,  1900,  No.  31. 

56 


STUDIES  ON  H^MOLYSINS. 


57 


If  we  bear  in  mind  that  the  toxins  in  a  restricted  sense  (diph- 
theria toxin,  tetanus  toxin,  etc.)  are  characterized  by  two  different 
groups,  of  which  one  is  haptophore  and  the  other  toxophore,  and 
if  we  express  this  by  means  of  a  diagram,  we  shall  find  that  the  anal- 
ogy between  toxins  and  haemolysins  becomes  very  apparent.  The 
active  hcemolysin  is  seen  to  be  nothing  but  a  toxin  consisting  of  two  parts. 
One  of  these  parts,  the  immune  body,  corresponds  to  the  haptophore 
group  of  the  toxin,  while  the  complement  represents  the  toxophore 

FIG.  l. 


o,  complement;    6,  interbody  (immune  body);    c,  receptor;    d,  part  of  a 
cell;    e,  toxophore  group  of  the  toxin;   /,  haptophore  group. 

group.1  In  opposition  to  our  views,  Bordet  assumes  that  the  immune 
body  (substance  sensibilatrice)  in  a  manner  not  definitely  stated, 
sensitizes  the  blood-cells  so  that  certain  injurious  substances  present 
in  normal  blood-serum  (alexins)  act  destructively  on  these  cells. 


1  This  analogy  becomes  apparent  also  in  heating,  for  the  toxins  as  well 
as  the  h£emolysins,  through  the  loss  of  the  toxophore  group  by  the  one,  or 
of  the  complement  (which  corresponds  to  the  toxophore  group)  by  the  other, 
lose  their  specific  action.  On  the  other  hand,  the  residues,  which  still  possess 
the  haptophore  group,  are  able  to  excite  the  production  of  specific  antibodies 
in  the  organism.  In  this  sense,  therefore,  the  toxoids  are  analogues  of  the  im- 
mune body. 


58  COLLECTED  STUDIES  IN   IMMUNITY. 

The  difference  between  these  two  views  is  considerable.  According 
to  our  views  the  complement  (  =  Bordet 's  alexin)  possesses  a  direct 
affinity,  due  to  chemical  relationship,  to  the  immune  body,  while 
according  to  Bordet  such  a  relation  is  excluded.  Since  this  question 
concerns  our  scientific  understanding  of  haemolysins  and  bacterioly- 
sins,  and  concerns  also  a  basic  difference  affecting  the  practical  appli- 
cation of  the  bacteriolysins,  we  shall  have  to  study  the  subject  more 
closely. 

I.    Concerning  Alexins. 

Buchner,  who  by  his  thorough  investigations  on  the  bactericidal 
and  globulicidal  properties  of  normal  sera  laid  the  most  important 
foundations  of  this  subject,  assumes  that  the  serum  contains  cer- 
tain protective  bodies,  alexins,  which  act  equally  on  bacteria,  foreign 
blood-cells,  etc.  These  alexins,  which  are  essentially  of  the  character 
of  proteolytic  enzymes,1  are  of  most  unstable  (labile)  nature  and  lose 
their  power  by  being  heated  to  55°  C.  Bordet  also  seems  to  assume 
the  presence,  in  normal  serum,  of  alexins  in  Buchner's  sense. 

According  to  Buchner,  the  serum  of  a  given  species  always  con- 
tains the  alexin  as  a  single  definite  substance.  Now  in  our  second 
communication  we  showed  that  the  matter  was  much  more  com- 
plicated than  this ;  that  in  the  hsemolysins  of  the  normal  sera  examined 
by  us  the  action  depends  on  the  combination  of  two  substances 
which  correspond  entirely  to  the  two  components  of  the  hsemolysin 
obtained  by  immunization.  Hence  an  "  alexin  "  also  consists  of 
an  interbody  which  withstands  heating,  and  a  complement  which 
is  generally  thermolabile'.2  The  interbody  is  in  every  respect  the 
complete  analogue  of  the  immune  body,  and  the  only  difference 
between  these  is  that  in  one  case  the  side-chains  of  the  protoplasm 
are  thrust  off  in  the  course  of  normal  vital  processes,  in  the  other 
case  this  is  due  to  an  immunizing  procedure. 

Since  our  second  communication  we  have  been  able  to  confirm 
this  view  by  means  of  a  large  number  of  separate  cases.  Of  these 
we  shall  mention  only  a  few  which  serve,  above  all,  to  support  the 
immediate  consequences  of  our  view,  namely,  the  multiplicity  of  the 
hcemolysins  of  normal  serum. 

Goat  serum  dissolves  the  blood-cells  of  rabbits  as  well  as  those 

1  Buchner,  Munch,  med.  Wochenschrift,  1900,  No.  9. 

2  Moxter  (Centralblatt  fur   Bacteriologie,   Vol.    26)    has  demonstrated  this 
also  for  a  normal  bacteriolysin. 


STUDIES  OX   H^MOLYSIXS.  59 

of  guinea-pigs.  Heating  the  serum  for  half  an  hour  to  55°  C. 
causes  this  property  to  be  lost,  owing  to  the  destruction  of  the  com- 
plements. On  the  other  hand,  one  frequently  finds  horse  sera,  by 
themselves  unable  to  dissolve  the  erythrocytes  of  rabbits  or  guinea- 
pigs,  which  are  able  through  their  content  of  complement  to  complete 
the  inactive  interbody  of  the  goat  serum  and  make  this  a  complete 
haemolysin.  According  to  Buchner's  views,  only  a  single  alexin  is 
concerned  in  haemolysis.  We  therefore  next  studied  the  question 
whether  the  interbodies  which  act  on  the  blood-cells  of  rabbits  and 
guinea-pigs  are  identical.  For  this  purpose  we  first  determined  the 
dose  of  inactive  goat  serum  which,  on  reactivation  by  the  addition 
of  sufficient  horse  serum,  was  able  to  dissolve  a  certain  amount  on 
rabbit  or  guinea-pig  blood-cells.  On  the  basis  of  these  data  this 
amount  of  rabbit  blood  in  physiological  salt  solution  was  mixed  with 
the  required  amount  of  inactive  goat  serum  and  after  standing  a 
short  time  at  room  temperature  the  mixture  was  centrifuged.  The 
result  was  as  follows:  The  clear  fluid  mixed  with  additional  rabbit 
blood  cells  and  the  activating  horse  serum  showed  no  trace  of  solvent 
property;  the  red  blood-cells,  originally  separated  by  centrifuging> 
dissolved  completely  under  the  influence  of  horse  serum.  In  a 
parallel  series  of  experiments  the  clear  fluid  was  mixed  with  guinea- 
pig  blood.  In  this,  complete  solution  ensued. 

From  these  experiments  the  conclusion  follows  that  rabbit  blood 
combines  with  an  interbody  present  in  goat  serum,  and  does  so, 
in  fact,  completely;  whereas  the  interbbdy  acting  on  guinea-pig  blood 
is  not  at  all  fixed  by  the  rabbit  blood.  By  means  of  this  elective 
absorption,  therefore,  it  is  positively  determined  that  normal  goat 
•serum  contains  two  interbodies,  one  acting  on  rabbit  blood  and  the 
other  on  guinea-pig  blood. 

The  question  at  once  arose  whether  these  interbodies  possess 
a  single  complement  in  common  or  whether  there  is  a  special  comple- 
ment for  each.  Only  after  considerable  labor  were  we  able  to  decide 
this  question  experimentally.  We  were  finally  able  to  determine 
that  in  the  filtration  of  normal  goat  serum  through  Pukall  filters, 
the  first  portion  (6-10  cc.)  possesses  a  markedly  different  solvent 
power  for  rabbit  and  guinea-pig  blood.  We  herewith  reproduce 
an  experiment  of  this  kind. 

0.15  cc.  of  goat-serum  previous  to  filtration  was  able  to  dissolve 
completely  2  cc.  of  a  5%  mixture  of  guinea-pig  blood,  while  0.2  cc. 
serum  was  able  to  dissolve  the  same  amount  of  rabbit  blood.  After 


60  COLLECTED  STUDIES  IN  IMMUNITY. 

the  serum  was  filtered,  the  filtrate  showed  the  same  solvent  power 
for  guinea-pig  blood,  whereas  the  solvent  power  for  rabbit  blood 
had  almost  entirely  disappeared,  for  0.8  cc.  effected  only  a  trace  of 
solution  and  0.23  cc.  none  at  all.  This  loss  of  solvent  power  could 
be  due  only  to  an  absorption,  by  the  filter,  of  (1)  the  interbody 
fitting  the  rabbit  blood,  or  (2)  the  complement,  or  (3)  both.  Since, 
however,  the  solvent  action  of  the  filtrate  on  rabbit  blood  was  restored 
by  the  addition  of  complement-containing  horse  serum,  while  the 
addition  of  interbody  had  no  effect,  it  follows  that  the  filtration 
had  removed  only  the  complement.  From  this  fact,  namely  that 
a  serum  may  be  deprived  of  its  complement  for  rabbit  blood  while 
the  complement  for  guinea-pig  blood  remains,  we  must  conclude 
that  there  are  two  different  complements  corresponding  to  these  two 
interbodies.  According  to  this,  then,  at  least  four  different  substances 
are  concerned  in  the  case  in  question,  two  different  immune  bodies 
and  two  complements  fitting  thereto.  One  pair  of  these  acts  on 
guinea-pig  blood  and  the  other  on  rabbit  blood.  According  to 
Buchner  only  one  single  substance,  the  alexin  of  goat  serum,  would 
be  concerned.  Further  details  of  these  experiments  will  be  published 
later.  We  should,  however,  like  to  observe  that  in  the  horse  serum 
used  for  reactivating,  it  was  possible  to  prove  the  existence  of  two 
complements.  This  proof,  moreover,  was  effected  in  two  ways, 
by  means  of  filtration  and  by  the  production  of  anticomplements. 
The  following  observation  will  show  that  a  still  greater  multi- 
plicity of  normal  hsemolysins  can  exist  in  the  serum.  In  our  second 
communication  we  have  given  a  detailed  description  of  an  experi- 
ment in  which  a  normal  interbody  of  dog  serum  was  caused  to 
combine  with  guinea-pig  blood  and  then  reactivated  by  means  of 
guinea-pig  serum,  which  served  to  supply  the  complement.  In  this 
experiment  the  interbody  contained  in  0.2  cc.  dog  serum  was  bound 
by  a  certain  quantity  of  guinea-pig  blood-cells.  This  is  the  amount 
of  dog  serum  which,  when  active,  just  suffices  to  completely  dissolve 
the  given  quantity  of  blood.  On  repeating  this  experiment,  but 
employing  horse  serum  as  complement,  it  was  found  impossible  to 
reactivate  the  dose  of  interbody  just  sufficient  for  solution  (0.2  cc.). 
By  systematic  trials,  in  which  multiples  of  the  dose  of  interbody 
previously  used  were  employed,  we  finally  determined  that  it  required 
six  times  the  amount,  i.e.,  1.2  cc.,  in  order  that  the  interbody  would 
be  completely  reactivated  by  the  horse  serum.  That  is,  the  first, 
employed  dose  of  the  inactive  dog  serum,  which  contained  just  sufficient 


STUDIES  OX   H.EMOLYSINS.  61 

interbody  to  be  completely  activated  when  the  complement  of  guinea- 
pig  serum  was  used,  contained  only  one-sixth  the  amount  of  interbody 
which  was  completely  activated  when  horse  serum  was  used  as 
complement.  From  this,  however,  it  follows  that  all  the  interbody 
present  hi  dog-serum  and  possessing  specific  relations  to  the  guinea- 
pig  blood-cells  is  not  of  the  same  uniform  nature.  In  our  case  one- 
sixth  of  the  interbody  acting  on  guinea-pig  blood  can  be  reactivated 
by  horse  serum,  while  fully  five-sixths  can  be  reactivated  by  the 
complement  of  guinea-pig  serum.  Therefore  the  goat  serum  con- 
tarns  two  different  interbodies  for  the  same  species  of  blood-cells,  and 
these  can  be  positively  separated  by  means  of  the  difference  in  activa- 
tion. 

In  our  second  communication,  by  showing  the  existence  of  a 
thermostabile  and  a  thermolabile  complement  in  the  goat  serum, 
we  also  proved  that  the  complements  of  a  given  serum  need  not 
be  of  uniform  nature.  At  that  time  we  showed  that  the  sera  of 
two  bucks  treated  with  sheep  blood-cells,  as  well  as  the  sera  of  a 
number  of  normal  goats,  contained  a  complement  which,  hi  con- 
trast to  the  other  complements  of  the  same  sera  (for  rabbit  blood 
and  guinea-pig  blood),  was  not  destroyed  by  heating  to  56°  C. 
Buchner  finds  it  so  hard  to  emancipate  himself  from  his  views  that  he 
seeks  to  explain  our  observations  by  assuming  we  made  a  gross  error 
in  the  experiment.  He  supposes  that  the  sheep  serum  still  present 
hi  the  5%  mixture  of  sheep  blood-cells,  and  which  we  disregarded, 
reactivated  the  inactive  serum  and  led  us  to  mistake  it  for  a  resistant 
complement.  We  were  well  aware  of  this  source  of  error  and  had 
therefore,  even  hi  the  first  communication,  stated  that  the  slight 
amounts  of  sheep  serum  present  in  the  blood  mixture  caused  no  dis- 
turbances whatever.  How,  by  the  way,  could  it  be  explained  that 
these  disturbances  occurred  only  in  the  serum  of  certain  animals 
although  the  method  of  procedure  was  the  same?  Or,  that  digestion 
of  the  serum  with  HC1,  which  does  not  injure  the  immune  body,  pre- 
vented all  solution  whatever? 

After  what  has  been  said,  we  shall  have  to  assume  that  in  gen- 
eral every  serum  which  acts  hcemolytically  on  various  species  of  blood 
possesses  a  corresponding  multiplicity  of  interbodies,  to  which  again 
different  complements  may  fit.  Against  the  Unitarian  views  of 
Buchner  and  of  Bordet  we  must  uphold  the  view  that  the  experi- 
mental results  positively  show  a  multiplicity  of  complements  in 
normal  serum.  This  multiplicity  of  the  haemolytic  substances  will 


62  COLLECTED  STUDIES  IN  IMMUNITY. 

not  be  surprising  if  we  remember  that  normal  blood  serum  con- 
tains, besides  the  hsemolysins,  a  number  of  other  active  substances 
such  as  hsemagglutinins,  bacterioagglutinins,  antiferments,  ferments, 
cytotoxins,  etc. ;  and  further,  that  from  a  normal  serum  which  agglu- 
tinates several  species  of  bacteria,  the  corresponding  agglutinin 
can  be  isolated  and  abstracted  by  treating  the  serum  with  one  of 
these  species  (Bordet);  and  that  the  same  holds  true  f or  hsemagglu- 
tinins  (Malkoff).  We  shall  quite  naturally  come  to  the  conclu- 
sion that,  under  normal  conditions  of  the  cell's  nutrition,  a  large 
number  of  simple  or  complex  side-chains  are  constantly  thrust  off 
which  then,  either  alone  or  in  conjunction  with  complements  simi- 
larly thrust  off,  exert  specific  actions.  Hence  normal  serum  contains 
an  enormous  number  of  such  substances.  To  these,  in  general,  we 
give  the  name  haptins. 

When  therefore  Buchner,  in  opposition  to  our  views,  believes 
that  the  assumption  of  these  different  substances  seems  unreasonable, 
we  must  emphasize  that  our  conclusions  are  not  the  result  of  specu- 
lation, but  simply  the  necessary  consequences  of  observations  which 
are  not  to  be  harmonized  with  the  assumption  of  a  single  simple 
alexin.  It  will  be  evident  also  why  we  have  completely  dropped 
the  term  alexin  used  by  Buchner.  In  our  investigations,  in  all  the 
cases  closely  analyzed,  we  never  found  a  simple  substance  (Buchner 's 
alexin) ,  but  always  a  complex  hsemolysin  consisting  of  interbody  and 
complement.  This  hsemolysin,  as  alreaady  emphasized,  completely 
corresponds  in  its  properties  to  the  hsemolysins  developed  through 
immunization.  We  shall  therefore  have  to  assume  that  also  in 
their  development  the  normal  hsemolysins  correspond  exactly  to  the 
artificial  hsemolysins. 

In  regard  to  the  latter,  von  Dungern  has  already  shown,  by 
demonstrating  a  great  disproportion  between  immune  body  and 
complement,  that  these  two  substances  are  produced  quite  inde- 
pendently of  one  another,  and  that  they  therefore  probably  originate 
in  different  cell  domains,  von  Dungern  also  showed  that  in  the 
extensive  formation  of  new  immune  body  which  occurred  when 
rabbits  were  treated  with  cattle  blood-cells,  the  corresponding  com- 
plement was  not  in  the  least  increased.  We  ourselves  have  often 
noted  an  analogous  independence  of  the  two  components  in  a 
number  of  normal  hsemolysins.  One  of  us  will  discuss  this  at  length 
in  a  subsequent  paper.  One  interesting  fact,  however,  we  shall  men- 
tion here. 


STUDIES  OX  HJEMOLYSIXS.  63 

If  rabbits  are  poisoned  with  a  dose  of  phosphorus,  of  which  they 
die  on  the  third  day,  and  if  the  serum  of  the  animal  is  collected  on 
the  second  day,  it  will  be  found  that  the  serum  has  lost  the  property, 
previously  possessed,  to  dissolve  guinea-pig  blood.  This  inactive 
serum  can  be  activated  by  the  addition  of  a  sufficient  amount  of 
guinea-pig  serum.  It  behaves,  therefore,  like  a  serum  which  has 
been  inactivated  by  heating  to  55°  C.,  i.e.  it  has  been  deprived  of 
its  complement.  It  is  probable  that  the  phosphorus  has  acted 
especially  on  certain  cell  domains  which  furnish  the  complements 
in  question. 

II.    Concerning  Anticomplements. 

In  accordance  with  the  views  already  discussed  in  detail,  we 
assume  that  the  hsemolytic  action  is  due  to  this,  that  the  interbody 
(immune  body)  and  complement  unite  to  form  the  complex  hsemolysin. 
We  can  understand  such  relations  only  when  we  regard  them  stereo- 
chemically  and  must  therefore  assume  that  the  complement  possesses 
a  haptophore  group  which  finds  in  the  interbody  a  receptor  group 
into  which  it  exactly  fits.  "With  this  conception,  however,  the  rela- 
tions existing  between  interbody  and  complement  at  once  assume 
a  strictly  specific  character,  i.e.,  the  interbody  and  complement  become 
strongly  specifically  related.  As  a  result  of  combining  experiments 
we  have  already  1  attacked  the  view  of  Bordet,  that  the  immune  body 
merely  sensitizes  the  red  blood-cells  and  that  as  a  result  of  this  sen- 
sitization  the  alexins,  which  otherwise  are  unable  to  attack  the  blood- 
cells,  now  have  access  to  them.  That  the  "  substance  sensibila- 
trice  "  breaks  the  way  for  the  alexins  is  a  coarse  mechanical  con- 
ception hardly  comprehensible  when  viewed  chemically  or  biologic- 
ally. If  one  sought  to  explain  Bordet's  view  chemically,  one  would 
have  to  assume  that  the  nature  of  the  sensitization  is  this,  that  under 
the  influence  of  the  sensitizor  a  whole  series  of  groups  are  developed 
in  the  protoplasm  of  the  red  blood-cells  which  are  able  to  bind  the 
various  complements.  Such  an  assumption,  however,  lacks  every 
element  of  probability.  Bordet2  himself  arrives  at  a  contradiction 
when  on  the  one  hand  he  assumes  a  direct  action  of  the  comple- 
ments on  the  red  blood-cells  and  on  the  other  is  forced  to  admit 
that  certain  relations  exist  between  interbody  and  complement 

1  See  our  second  communication. 

1  Bordet,  Annales  de  1'lnstitut  Pasteur,  May  1900. 


<54  COLLECTED  STUDIES  IN  IMMUNITY. 

(certains  rapports  convenables).  It  would  be  difficult  to  express 
these  relations  in  a  form  chemically  comprehensible. 

Based  on  the  conception  of  strictly  specific  relations,  such  as 
follows  from  our  theory,  the  study  of  these  complements  acquires  a 
high  practical  value.  Donitz  1  has  already  called  attention  to  the 
great  importance  for  the  therapy  of  infectious  diseases  of  finding 
sources  yielding  sufficient  complement,  von  Dungern2  has  further- 
more shown  that  body  cells  are  able  to  bind  certain  complements  and 
that  therefore  a  completed  bacteriolysin  derived  from  a  certain 
animal  species  can,  when  it  is  injected  into  another  organism,  entirely 
lose  its  complement  and  so  become  inactive. 

In  the  Croonian  lecture  (March  22,  1900),  Ehrlich  pointed  out 
that  the  bacteriolysins  and  hsemolysins  (interbody + complement) 
possess  three  haptophore  groups,  of  which  two  are  on  the  interbody 
and  one  on  the  complement.  It  is  conceivable  that  for  each  of 
these  groups  there  is  a  corresponding  antigroup  which  binds  the 
haptophore  concerned  and  so  inhibits  the  action  of  the  lysin.  For 
each  lysin  therefore  three  antibodies  are  possible,  the  action  of  any 
one  of  which  is  able  to  put  the  lysin  out  of  action.  At  that  time 
Ehrlich  called  particular  attention  to  the  important  role  of  one  of 
these  antibodies,  namely,  the  one  which  fits  into  the  haptophore 
group  of  the  complement  and  so  prevents  this  from  combining  with 
the  interbody  (immune  body).  He  stated  further  that  together 
with  Morgenroth  he  had  succeeded  in  the  experimental  production 
of  such  anticomplements  by  means  of  immunization.3 

Our  observations  in  this  direction  were  made  on  the  serum  of 
a  goat  which  for  a  long  time  had  been  injected  with  large  amounts 
of  horse  serum.  Horse  serum  was  used  because  our  extended  ob- 
servations had  shown  that  this  constitutes  a  particularly  rich  source 
of  most  varied  complements,  and  because  it  was  therefore  to  be 
expected  that  a  plentiful  amount  of  anticomplements  would  be 
obtained.  This  expectation  was  fully  realized,  and  we  have  come 
to  know  a  large  number  of  interbodies  of  different  origin  which  can 
be  reactivated  by  the  complements  for  different  varieties  of  blood 
contained  in  horse  serum.  As  an  example  the  following  combina- 
tions may  be  mentioned.:  Rabbit  blood — inactive  dog  serum;  guinea- 

1  Donitz,  Klinisches  Jahrbuch,  Vol.  7,  1899. 

2  See  page  36. 

3  In  the  meantime  Bordet  (loc.  cit.)  independently  has  also  produced  anti- 
complements  by  means  of  immunization. 


STUDIES  OX  H.EMOLYSINS.  65 

pig  blood — inactive  goat  serum;  sheep  blood — inactive  dog  serum; 
sheep  blood  and  inactive  serum  of  goats  treated  with  sheep  blood. 
In  all  these  cases  we  have  been  able  to  determine  that  the  reactivat- 
ing action  of  the  horse  serum  can  be  prevented  by  the  addition  of 
small  amounts  of  anticomplement  serum  (previously  inactivated). 

In  one  case  a  very  minute  analysis  of  this  action  was  made.  The 
factors  in  this  case  were  rabbit  blood  and  an  interbody  acting  on 
this,  present  in  normal  goat  serum  and  obtained  by  heating  the 
serum  to  56°  C.  The  rabbit  erythrocytes  were  first  treated  with 
considerable  amounts  of  this  interbody  and  the  excess  of  inter- 
body  was  then  separated  by  centrifuging  the  mixture  and  pouring  off 
the  clear  fluid.  The  erythrocytes  thus  loaded  with  interbody  were 
next  digested  with  large  amounts  of  the  inactive  anticomplement 
serum  and  this  likewise  separated  by  centrifuging.  The  sedimented 
blood-cells  thus  obtained  dissolved  completely  on  the  addition  of 
horse  serum.  The  same  result  was  attained  when  the  process  just 
described  was  performed  in  one  act  instead  of  in  two;  i.e.,  by  mixing 
the  goat  serum  containing  the  interbody  with  the  anticomplement 
serum  before  the  addition  of  the  blood-cells. 

From  this  it  follows  that  the  antibody  stands  in  relation  neither 
to  the  blood-cells  themselves  nor  to  the  interbody.  Even  in  the  pres- 
ence of  the  antibody  the  interbody  is  anchored  in  normal  fashion  by 
the  erythrocytes,  and  is  furthermore  not  disturbed  in  its  receptive 
property  for  the  complement.  The  antibody  therefore  has  no 
relation  to  either  of  the  two  haptophore  groups  of  the  interbody,  and 
it  can  only  act  by  influencing  the  complement. 

The  complement,  however,  according  to  our  view,  also  possesses 
two  groups:  one,  a  haptophore  group,  and  a  second  which,  in  order 
to  express  the  analogy  to  the  enzymes  and  toxins,  we  shall  term  the 
zymotoxic  group.  Hence  it  still  remained  to  determine  into  which 
of  these  two  groups  the  anticomplement  fits.  In  either  case,  though 
of  course  by  a  different  mechanism,  the  action  of  the  complement 
would  be  inhibited;  in  one  case  by  preventing  the  combination  of 
complement  and  interbody,  in  the  other  by  preventing  the  zymo- 
toxic action. 

If  we  assume  that  the  anticomplement  combines  with  the  zymo- 
toxic group,  then  the  haptophore  group  of  the  complement  will  remain 
free  and  must  still  be  able  to  combine  with  the  corresponding  group 
of  the  interbody.  It  would  be  expected,  then,  that  the  haptophore 
group  would  combine  with  the  interbody  and  "plug,"  so  to  speak, 


66  COLLECTED  STUDIES  IN  IMMUNITY. 

the  binding  group  of  the  latter  against  any  further  combination  with 
complement.  If,  on  the  contrary,  the  anticomplement  combines 
with  the  haptophore  group  of  the  complement,  the  interbody  is 
left  free  and  must  therefore  still  be  capable  of  reactivation.  The 
experimental  solution  of  this  question  was  very  easy.  The  erythro- 
cytes,  loaded  with  interbody,  were  subjected  to  the  action  of  a 
mixture  of  complement  and  anticomplement  which  had  been  neu- 
tralized to  complete  inactivity.  After  centrifuging  it  was  found  that 
the  blood-cells  dissolved  readily  on  the  further  addition  of  comple- 
ment. Solution  also  occurs  if  a  small  amount  of  complement  in 
excess  is  added  to  the  exactly  balanced  mixture  of  complement  and 
anticomplement.  These  experiments  indicate  that  the  anticomple- 
ment acts  by  fitting  into  the  haptophore  group  of  the  complement  and 
side-tracking  this  group. 

We  have  also  convinced  ourselves  that  it  is  possible  to  produce 
anticomplements  not  only  with  horse  serum  but  also  with  other 
sera,  such  as  the  sera  of  goats,  dogs,  cattle,  rabbits,  and  guinea-pigs, 
by  injecting  the  serum  into  foreign  species.  In  these  experiments  the 
choice  of  animals  employed  for  purposes  of  immunization  also  plays 
an  important  role.  For  example,  a  rabbit  treated  with  goat  serum 
very  readily  yields  an  anticomplement,  whereas  when  a  dog  was 
similarly  injected  no  anticomplement  (at  least  in  the  two  cases 
examined  by  us)  could  be  demonstrated.  So  far  as  we  were  able 
to  determine,  the  protection  afforded  by  the  anticomplement  extends 
to  all  the  species  of  blood-cells  on  which  the  serum  used  for  immuni- 
zation exerts  its  action.  Since  the  sera  in  question,  so  far  as  lysin 
action  is  concerned,  contain  a  plurality  of  complements,  the  anti- 
complementary  serum  must  contain  a  whole  series  of  anticomple- 
ments which  correspond  to  the  different  complements  present  in 
the  immunizing  serum.  Perhaps  this  polyvalence  of  the  anticom- 
plementaiy  serum  accounts  for  the  phenomenon  that  certain  anti- 
sera  produced  by  means  of  a  particular  blood  serum,  are  able  to 
inhibit  the  injurious  action  of  many  other  kinds  of  blood  serum. 
These  facts  indicate  that  this  interchange  of  protection  is  due  to 
the  presence  in  the  two  sera  of  a  certain  number  of  common  com- 
plements, In  fact  there  seem  to  be  cases  in  which  certain  species 
have  the  majority  of  their  complements  similar.  Such  a  case  in 
all  probability  is  that  of  the  goat  and  the  sheep,  as  is  evidenced  by 
the  fact  that  in  the  reactivating  action  goat  serum  can  be  completely 
replaced  by  sheep  serum  and  vice  versa.  This  at  least  is  true  for 


STUDIES  ON  ILEMOLYSIXS.  67 

all  the  cases  observed  by  us.  Still  more  convincing,  however,  is  the 
fact  that  neither  the  injection  of  a  sheep  with  goat  serum  nor  of  a 
goat  with  sheep  serum  results  in  the  production  of  anticomplements. 
All  experiences  indicate  that  the  complements  normally  present  in 
the  serum  of  a  certain  species  of  animal  are  not  able  to  excite  the 
formation  of  anticomplements  in  such  an  animal's  own  body.  Per- 
haps this  may  be  explained  thus,  that  the  relation  between  com- 
plement and  complementophile  group  is  extremely  slight  (as  was 
shown  by  the  binding  experiments  previously  described  by  us)  and  that 
therefore  one  of  the  conditions  necessary  for  the  thrusting  off — a  per- 
manent and  firm  union  with  the  receptor — is  not  in  this  case  fulfilled. 
We  realize  that  we  have  been  able  here  merely  to  point  out  some 
of  the  principles  applying  to  this  subject.  Their  closer  anatysis 
encounters  extraordinary  difficulties  in  consequence  of  one  of  the 
facts  demonstrated  by  us,  namely,  the  multiplicity  of  interbodies, 
complements,  and  anticomplements.  Thus  far  these  difficulties  have 
been  overcome  in  only  a  few  favorable  instances. 

HI.    One  of  Bordet's  Objections  Controverted. 

Bordet,  in  his  most  recent  work  (loc.  cit.)  has  described  the  follow- 
ing interesting  experiment,  by  means  of  which  he  believes  to  prove 
that  our  views  concerning  the  mechanism  of  haemolysis  are  incor- 
rect. As  hsmolysin,  Bordet  employed  the  serum  of  guinea-pigs  after 
these  had  been  treated  with  rabbit  blood.  This  then  possessed  a 
high  degree  of  solvent  power  for  rabbit  blood.  If  this  haemolysin 
is  inactiviated  by  heating,  it  is  possible  to  restore  the  haemolytic 
action,  as  well  by  the  addition  of  normal  guinea-pig  serum  as  by 
that  of  normal  rabbit  serum.  These  two  sera,  therefore,  contain 
complements  (alexins)  which  make  the  reactivation  possible.  Bordet 
now  sought  to  discover  whether  the  "alexin"  of  rabbits  is  identical 
with  that  of  guinea-pigs.  For  this  purpose  he  treated  rabbits  with 
the  serum  of  the  immunized  guinea-pigs  and  obtained  an  antiserum 
which,  while  it  contained  a  small  amount  of  anti-immune  body, 
contained  considerable  anticomplement.  He  then  determined  that 
this  "antialexin"  acted  only  against  the  "alexin"  of  the  guinea- 
pig  and  not  at  all  against  that  of  rabbits  and  some  other  animals. 
At  the  same  time  a  certain  degree  of  action  against  the  complement 
of  pigeon  serum  was  noted,  so  that  this  antiserum  was  not  absolutely 
specific.  From  this  Bordet  concludes  that  his  theory  of  sensitiza- 
tion  must  be  correct,  namely,  that  the  various  alexins  derived  from 


68  COLLECTED  STUDIES  IN  IMMUNITY. 

different  species  act  directly  injuriously  on  the  sensitized  blood-cells. 
Against  each  of  these  alexins  an  antialexin  exists  which  protects 
the  sensitized  blood-cells  against  just  this  particular  alexin. 

It  cannot  be  denied  that  at  first  sight  this  experiment  appears 
to  speak  strongly  in  favor  of  Bordet's  theory.  If  one  assumes,  as 
Bordet  of  course  does,  that  in  the  immune  serum  produced  by  him, 
one  single  immune  body  comes  into  play,  then  since  this  can  be  reac- 
tivated as  well  by  rabbit  serum  as  by  guinea-pig  serum,  the  com- 
plement contained  in  these  two  species  of  sera  must,  according  to 
our  theory,  possess  the  same  haptophore  group.  If  this  were  the 
case,  however,  the  same  anticomplement  should  protect  against  both 
complements,  and  this  it  does  not  do. 

We  have  therefore  subjected  Bordet's  experiment  to  an  exact 
reexamination  and  have  been  able  to  determine  that  an  exhaustive 
quantitative  analysis  presents  the  experiment  in  an  entirely  different 
light.  A  hsemolytic  serum  was  produced  by  treating  guinea-pigs 
with  rabbit  blood.  A  preliminary  trial  of  this  serum  showed  that 
when  inactivated  it  could  be  reactivated  in  large  amounts  as  well 
by  guinea-pig  serum  as  by  rabbit  serum.  The  anticomplement, 
derived  from  other  rabbits  by  treatment  with  normal  guinea-pig 
serum,1  was  able  in  the  inactive  state  to  completely  inhibit  the  reacti- 
vation with  guinea-pig  serum,  although  the  same  anticomplement 
serum  in  its  active  state  reactivated  the  inactive  immune  body. 

We  next  proceeded  to  examine  these  facts  quantitatively  and 
found  that  the  simple  solvent  dose  of  the  serum  for  0.5  cc.  of  a  5% 
rabbit-blood  mixture  amounted  to  0.075  cc.  Then  we  tried  von 
Dungern's  experiment  (loc.  cit.)  to  increase  this  action,  by  adding 
to  the  native  immune  serum  normal  guinea-pig  serum  in  amounts  so 
small  that  they  did  not  themselves  exert  any  solvent  action.  We 
found  that  the  full  solvent  dose  had  thus  been  decreased  to  0.025  cc. 
This  proved,  as  in  von  Dungern's  case,  that  in  the  immunization  a 
large  excess  of  free  immune  body  was  present  which  could  not  nearly 
be  satisfied  by  the  amount  of  complement  normally  present.  Now 
we  could  expect  that  this  same  increase  in  power  would  be  effected 
bv  the  addition  of  rabbit  serum,  but  we  found  instead  that  rabbit 
serum  even  in  large  amounts  did  not  produce  any  increase  whatever. 

According  to  Bordet's  view  such  a  deviation  is  absolutely  incom- 
prehensible, and  this  led  us  to  pursue  the  case  further.  We  first 

1  In  contrast  to  Bordet  we  chose  normal  guinea-pig  serum  for  immunization 
in  order  to  avoid  the  disturbing  action  of  an  immune  body. 


STUDIES  ON  H^MOLYSINS.  69 

inactivated  the  immune  serum  and  determined  the  minimal  amount 
of  the  inactive  serum  which  would  cause  complete  solution  in  the 
presence  of  (1)  normal  rabbit  serum,  or  (2)  of  guinea-pig  serum. 
We  found  that  it  required  0.25  cc.  of  the  inactive  immune  serum  to 
effect  complete  solution  of  the  given  amount  of  rabbit  blood  when 
rabbit  complement  was  employed,  whereas  only  0.025  cc.  of  the  immune 
serum  was  required  when  guinea-pig  complement  was  employed. 

This  result,  however,  cannot  be  harmonized  with  Bordet 's  theory  of 
sensitization.  According  to  his  view  one  would  expect  that  a  blood- 
cell  which  is  sensitized  by  the  presence  of  the  immune  body  is  subject 
equally  to  the  action  of  various  alexins.  In  both  cases  the  same 
amount  of  immune  body  should  then  suffice  to  make  the  blood-cells 
sensitive  to  the  alexins  (complements).  As  a  matter  of  fact,  how- 
ever, it  requires  ten  times  as  much  in  the  one  case  as  in  the  other. 
If  one  desired  to  hold  to  Bordet 's  theory  one  might  possibly  say  that  it 
requires  ten  times  as  strong  a  sensitization  with  the  same  immune  body 
in  order  to  make  the  cells  sensitive  to  the  alexin  of  rabbit  serum. 

If  this  highly  complicated  assumption  wrere  correct,  the  relation 
as  above  determined,  1  :  10,  should  represent  a  constant  ratio.  Owing 
to  a  lack  of  animal  material  we  were  unable  to  study  this  question 
of  constant  ratio  on  the  example  selected  by  Bordet.  However, 
in  an  analogous  series  of  cases  for  which  we  had  abundant  material, 
we  were  able  to  pursue  this  question  further. 

We  made  use  of  a  goat  which  had  been  treated  with  sheep  blood 
and  whose  serum  therefore  dissolved  sheep  blood-cells.  The  inac- 
tivated serum  of  this  goat  could  be  reactivated  by  two  complements, 
that  of  normal  goat  serum  and  that  of  horse  serum.  The  anticom- 
plement  obtained  by  treating  a  goat  with  horse  serum  inhibited, 
even  in  small  amounts,  the  action  of  the  horse  complement;  whereas 
its  action  on  the  goat  complement  was  so  slight  as  to  be  practically 
negligible.  The  conditions  here,  therefore,  are  exactly  the  same 
as  in  the  case  described  by  Bordet. 

In  the  beginning  of  the  observations  it  was  found  that  1  cc.  of 
a  5%  mixture  of  sheep-blood,  mixed  with  normal  horse  serum  to 
serve  as  complement,  was  completely  dissolved  on  the  addition  of 
0.35  cc.  immune  body  (inactivated  immune  serum);  whereas  when 
normal  goat  serum  was  used  as  complement  only  0.025  cc.  of  the 
immune  body  was  required.  This  corresponds  to  a  ratio  of  14  : 1. 
On  repeating  the  test  a  week  later  with  serum  freshly  drawn  from 
the  immunized  goat  we  found  that  the  constituents  which  were 


70  COLLECTED  STUDIES  IN  IMMUNITY. 

reactivated  by  horse  serum  were  unchanged  (0.35),  but  that  it  required 
considerably  more  immune  body  when  goat  serum  was  used  for 
reactivation  than  it  had  before,  namely,  0.1  cc.  This  corresponds 
to  a  ratio  of  3.5  : 1  as  compared  to  the  former  ratio  of  14  : 1.  This 
shows  that  a  constant  ratio  does  not  as  a  matter  of  fact  exist.  We 
must  rather  assume,  as  we  did  for  a  normal  hsemolytic  serum,  that 
two  entirely  independent  immune  bodies,  A  and  B,  are  present  in 
the  immune  serum  and  that  these  differ  in  the  ratio  of  their  quan- 
tities and  in  the  manner  in  which  they  are  reactivated.  The  amount 
of  immune  body  A  contained  in  the  immune  serum  has  remained 
constant,  while  B  after  a  short  time  has  considerably  decreased 
(to  one  quarter).  This  divergence  would  in  fact  indicate  that  the 
two  immune  bodies  are  formed  independently  of  each  other. 

We  have  thus  demonstrated  that  in  the  phenomenon  observed 
,by  Bordet  not  a  single  immune  body,  but  two  different  ones,  come 
into  play,  one  of  which  is  related  to  a  complement  found  only  in 
guinea-pig  serum,  while  the  other  is  related  to  a  complement  found 
in  rabbit  serum.  Through  this  demonstration  Bordet 's  objection 
loses  all  its  force  and  his  experiment  becomes  in  fact  a  new  argu- 
ment for  our  theory. 

The  occurrence  of  different  immune  bodies  in  a  haemolytic  serum 
obtained  by  immunizing  with  red  blood-cells  is  not  at  all  surprising 
in  view  of  our  experiments  on  isolysins  described  in  our  third  com- 
munication. We  have  obtained  a  whole  series  of  different  isolysins 
by  injecting  goats  with  goat  blood.  .  At  present  they  number  twelve. 
In  the  red  blood-cells  not  merely  a  single  group  but  a  large  number 
of  different  groups  must  be  considered,  which,  provided  there  are 
fitting  receptors,  can  produce  a  corresponding  series  of  immune 
bodies.  All  of  these  immune  bodies  again  will  be  anchored  by  the 
blood-cells  employed  in  immunization.  We  may  assume  that  when 
an  animal  species  A  is  immunized  with  blood-cells  of  species  B  a 
haemolytic  serum  will  be  produced  which  contains  a  great  host  of 
immune  bodies.  These  immune  bodies  in  their  entirety  are  anchored 
by  the  blood-cells  of  species  A. 

We  are  convinced  that  the  duality  found  by  us  in  the  two  cases 
examined  is  much  below  the  actuality,  and  that  thorough,  though 
to  be  sure  arduous,  studies  will  succeed  in  discovering  a  multiplicity 
heretofore  unexpected.  For  the  present,  however,  this  duality  of 
the  immune  body  should  suffice  to  controvert  the  objections  made 
by  Bordet  from  the  Unitarian  standpoint. 


VH.    STUDIES  ON  ILEMOLYSINS.1 

FIFTH  COMMUNICATION. 
By  Professor  Dr.  P.  EHRLICH  and  Dr.  J.  MORGENROTH. 

IN  the  few  years  since  its  formulation  the  side-chain  theory  has 
exercised  a  marked  influence  on  the  direction  of  the  investigations 
in  immunity.  The  subject  of  toxins  and  antitoxins-  has  to  a  certain 
extent  been  concluded,  at  least  for  the  present.  Several  objections 
raised  by  Roux  and  Borrel2  in  connection  with  their  splendid 
work  on  cerebral  tetanus,  as  well  as  those  made  by  Metchni- 
koff2  and  Marie,2  rested  on  a  misconception  of  the  theory,  and  the 
facts  on  which  these  are  based  serve  rather  as  a  complete  confirma- 
tion of  the  theory.3  The  attempt  of  Pohl4  to  place  the  doctrine 
of  antitoxins  purely  on  the  basis  of  inorganic  chemistry  has  been 
completely  controverted  by  Bashford.5 

Thus  the  facts  proved  themselves  thoroughly  in  harmony  with 
the  theory,  and  the  latter  furthermore  proved  its  inventive  value 
in  many  directions.  It  was  but  natural  that  the  side-chain  theory 
originally  formulated  for  the  antitoxins,  if  it  had  any  general 
biological  significance  at  all,  should  also  include  the  complicated 
phenomena  of  immunity  which  result  from  the  introduction  of 
bacteria  or  tissue-cells.  Hence  we  began  two  years  ago  to  investigate 
experimentally  the  applicability  of  the  doctrines  resulting  from  this 
theory  to  the  specific  hsemolysins  obtained  by  immunization,  which 
had  been  discovered  by  Bordet  a  short  time  previously.  These  studies 

1  Reprint  from  the  Berliner  klin.  Wochenschrift,  1901,  Xo.  10. 

2  Annales  de  Plnstitut  Pasteur,  1898. 

3  See  Weigert,  Lubarsch's  Ergebnisse  der  Pathologie,  1897;    also  Levaditi 
Press  medicale,  1900,  Xo.  95. 

4  Arch,  internat.  de  Pharmacodyn.,  1900. 

5  Arch,  interaat.  de  Pharmacodyn.  et  Therapie,  Vol.  VIII,  fasc.  I  and  EC, 
1901. 

71 


72  COLLECTED  STUDIES   IN  IMMUNITY. 

served  to  demonstrate  the  complete  harmony  of  the  theory  with  the 
facts  on  this  subject.     Furthermore  after  overcoming  considerable 
experimental  difficulties  we  succeeded  in  demonstrating  the  same 
behavior  for  the  hsemolysins  of  normal  serum  and  thus  brought  these 
also  under  the  laws  of  the  side-chain  theory.     Reexaminations  from 
various   directions   confirmed   the   correctness   of   our    fundamental 
experiments  and  we  may  say  that  at  present  the  majority  of  workers 
in  this  field,  partly  as  a  result  of  their  own  experiments,  have  accepted 
our  views  and  regard  the  side-chain  theory  as  a  justified  hypothesis 
which  best  explains  most  of  the  phenomena  thus  far  observed  in  the 
subject   of   immunity.     Since    this   in    part    concerns    processes    in 
which  the  animal  organism  acts  with  all  its  highly  complicated  con- 
ditions, it  is  no  wonder  that  now  and  then  a  fact  has  appeared  in 
the  course  of  the  investigations  which  at  first  seemed  to  be  irrecon- 
cilable with  the  theory.     The  latter,  however,  is  in  no  way  injured 
thereby,   for  the  solution  of  such   apparent   contradictions   results 
in  a  deeper  understanding  of  the  subject  and  makes  for  progress. 
An  instructive  example  of  this  was  recently  afforded  in  physical 
chemistry.     As  is  well  known,  several  at  first  inexplicable  contra- 
dictions to  van't  Hoff's  theory  of  solutions,  resulting  from  certain 
deviations  in  osmotic  tension,  found  their  explanation  in  the  theory 
of  electrolytic  dissociation  of  Arrhenius,  and  this  theory  served  to 
again  obtain  general  acceptance  for  the  theory  of  solutions  itself. 
We  have  therefore  endeavored  to  analyze  carefully  the  objections 
urged  against  our  views  by  high  authorities. 

The  objection  raised  by  Metchnikoff  1  against  the  specific  formation 
of  the  toxins  was  based  on  the  fact  that  even  castrated  rabbits  yield 
an  antispermotoxin.  In  a  recent  study  2  from  the  laboratory  of 
Metchnikoff,  this  objection  is  withdrawn.  It  was  found  that  in  this 
antispermotoxin  an  an ti complement  is  principally  concerned  and 
not  an  anti-immune  body,  for  it  was  produced  even  by  treatment 
with  normal  serum.3  It  is  therefore  especially  gratifying  that  Metch- 
nikoff also  has  recently  accepted  our  view  that  the  complement 
is  anchored  to  the  immune  body  by  means  of  the  latter's  complement- 
ophile  group. 

An  important  objection  made  by  Bordet  4  based  on  some  extremely 

1  Annales  de  1'Institut  Pasteur,  1900,  No.  1. 

2  Ibid.,  No.  9. 

3  See  von  Dungern,  page  47. 

4  Annales  de  PInstitut  Pasteur,  1900,  No.  5. 


STUDIES  OX   H.EMOLYSIXS.  73 

interesting  experiments,  by  which  he  believed  to  refute  our  theory 
of  the  mechanism  of  haemolysis,  has  been  discussed  by  us  in  our 
fourth  communication l  and  controverted  by  means  of  extended  quan- 
titative experiments. 

It  is  necessary,  however,  once  more  to  thoroughly  discuss  the 
binding  of  immune  body  to  the  erythrocyte,  for  on  this  point  the 
views  seem  not  at  all  clear,  because  the  purely  chemical  conception  is 
denied  by  some  authors  or  is  regarded  as  unimportant. 

I.    The  3Ianner  in  which  the  Immune  Body  Combines  with  the 

Erythrocytes. 

In  our  first  communication  we  had  already  shown  that  the  ery- 
throcytes  as  such  behave  quite  differently  toward  the  two  components 
which  effect  haemolysis.  The  blood-cells  abstract  the  immune  body 
from  its  medium  with  great  avidity,  whereas  they  do  not  take  up  the 
slightest  trace  of  complement.  When  loaded  with  immune  body, 
however,  they  are  able  to  anchor  the  complement  also.  From  this 
we  have  concluded  primarily  that  the  immune  body  possesses  two 
combining  groups  of  different  affinity,  of  which  the  one  combines  with 
a  corresponding  group,  the  receptor  of  the  blood-cell;  the  other  com- 
bines with  the  complement.  But  according  to  our  view  these  combi- 
nations are  pure  chemical  phenomena  proceeding  between  immune 
body  and  blood-cells  and  between  immune  body  and  complement. 

The  function  of  the  immune  body  can  be  elucidated  by  means  of 
a  chemical  example,  that  for  instance  afforded  by  the  behavior  of 
diazobenzaldehyd.  Through  its  diazo  group  this  substance  can 
unite  with  a  series  of  bodies,  especially  with  amines,  phenols,  keto- 
methylen  groups,  whilst  the  aldehyd  group  on  its  part  can  effect  a 
series  of  syntheses — e.g.  with  hydrazins,  hydrocyanic  acid,  etc.  It 
thus  becomes  easy  by  means  of  diazobenzaldehyd  to  effect  a  com- 
bination between  substances  which  by  themselves  do  not  combine, 
as  phenol  and  hydrocyanic  acid.  Such  a  combination  includes  both 
substances.  In  order  to  make  the  comparison  still  closer  let  us  imagine 
that  certain  constituents  of  the  living  cell,  say  by  means  of  an  aro- 
matic group,  are  able  to  unite  with  the  diazo  combination.  In 
this  case  it  follows  that  by  means  of  the  aldehyd  group  of  the  diazo- 
benzaldehyd a  second  highly  toxic  nucleus — e.g.  that  of  hydrocyanic 
acid — can  be  joined  to  the  combination  in  such  fashion  that  the  proto- 
plasmal  molecule  is  now  subjected  to  the  action  of  the  strongly 

1  See  page  56.     • 


74  COLLECTED  STUDIES  IN   IMMUNITY. 

acting  nitril  group.  In  this  schematic  example  the  diazo  group  which 
fits  directly  into  the  protoplasm  would  correspond  to  the  haptophore 
group  of  the  immune  body  which  fits  into  the  receptor  of  the  blood- 
cells;  the  aldehyd  remnant  would  correspond  to  the  complemento- 
phile  group  of  the  immune  body.  The  complement,  which  as  we 
know  possesses  toxic  properties,  would  then  be  compared  to  the  hydro- 
cyanic acid.1 

The  facts  described  by  us  have  been  confirmed  from  various  sides 
(v.  Dungern,  Buchner,  Bordet)  by  experiments  on  blood-cells.  Bor- 
det 2  and  also  Nolf  3  showed  that  the  stromata  of  the  blood-cells,  which 
represent  the  protoplasma,  effect  the  anchoring  of  the  immune  body, 
while  the  haemoglobin,  which  is  to  be  regarded  as  paraplasma,  takes 
no  part  whatever  in  this  binding.  This  fact  corresponds  entirely 
to  the  views  expressed  by  Ehrlich  in  an  earlier  study  on  blood-cell 
poisons.4  Furthermore,  it  has  been  shown  by  von  Dungern  5  that  the 
power  of  the  blood-cells  to  excite  a  specific  hamolysin  by  immuniza- 
tion can  be  entirely  inhibited  by  completely  loading  the  receptors 
of  the  blood-cells  with  the  immune  body  in  question.  These  addi- 
tional facts  were  well  fitted  to  still  further  support  the  chemical  con- 
ception of  these  processes. 

Now,  however,  Bordet  has  described  an  experiment  which  he 
believes  shows  that  the  fixation  of  the  immune  body  is  not  a  chemical 
process  in  the  strict  sense,  but  that  this  phenomenon  is  to  be 
classed  rather  with  surface  attraction  and  similar  actions,  and  that 
it  is  completely  analogous  to  staining  processes.  These  views  are  also 
shared  by  Nolf  6  and  Nicolle.7 

Bordet's  experiment  in  the  main  is  as  follows:  By  treating  a 
guinea-pig  with  rabbit  blood  a  haemolytic  serum  is  obtained  specific 
for  rabbit  blood-cells.  A  certain  amount  of  the  serum  dissolves  an 
absolutely  definite  amount  of  rabbit  blood-cells  if  all  the  cells  are 
added  to  the  serum  at  once.  If ,  however,  to  the  same  amount  of  serum 

1  One  could  designate  substances  which,  like  the  immune  bodies,  are  supplied 
with  two  different  combining  groups  as  amboceptors.     This  name  would  indicate 
the  double  binding  function  as  well  as  the  fact  that  they  correspond  to  thrust- 
off  receptors. 

2  Loc.  cit. 

3  Annal.  de  1'Institut  Pasteur,  1900. 

4  Charite  Annalen,  Vol.  X. 

5  See  page  36. 

6  Loc.  cit. 

7  Revue  generate  des  Materies  Colorantes,  1900,  Nos.  43  and  44. 


STUDIES  OX  H^MOLYSINS.  75 

only  one-half  the  amount  of  blood-cells  is  first  added,  sufficient  time 
allowed  for  these  to  completely  dissolve  and  the  second  half  of  the 
blood-cells  added,  it  will  be  found  that  these  are  no  longer  dissolved. 
It  appears,  therefore,  as  though  the  blood-cells  were  capable  of  com- 
bining with  double  the  amount  of  immune  body  necessary  for  their 
solution.  In  order  to  explain  this  result  Bordet  describes  the  follow- 
ing staining  experiment:  If  one  dissolves  methyl  violet  in  water,  it 
is  possible,  by  means  of  a  strip  of  filter-paper  dipped  into  the  solution, 
to  abstract  all  the  coloring-matter  from  the  solution.  The  strip  will 
assume  a  color  of  very  definite  intensity.  If,  however,  the  strip  is 
divided  into  several  smaller  strips  and  these  are  dipped  into  the 
fluid  one  after  the  other,  the  first  strip  will  assume  a  considerably  deeper 
color,  whereas  the  strips  last  introduced  will  be  unable  to  abstract 
any  color  from  the  now  colorless  fluid.  From  this  Bordet  draws 
the  following  conclusion: 

"On  peut  admettre,  par  comparaison,  que  les  premiers  globules 
introduits  dans  Phemotoxine  sont  deja  susceptibles  de  perdre  leur 
hemoglobine  lorsqu'ils  ne  sont  encore  que  "  faiblements  teints  "  par 
les  principes  actifs,  mais  qu'ulterieurement  ils  peuvent  absorber  une 
dose  beaucoup  plus  grande  de  ces  substances,  epuiser  ainsi  le  serum 
et  empecher  la  destruction  de  nouveaux  globules  introduits  dans  la 
suite." 

Phenomena  such  as  those  here  described  have  long  manifested 
themselves  in  our  experiments  on  the  binding  of  the  immune  body 
by  the  erythrocytes  although  these  experiments  were  of  somewhat 
different  form.  But  before  we  proceed  to  discuss  these  results  and 
our  conclusions,  we  should  like  to  describe  the  facts  observed  by  us. 

In  order  to  determine  the  combining  ability  of  the  erythrocytes 
for  an  immune  body,  especially  when  quantitatively  accurate  results 
are  desired,  it  is  best  to  proceed  as  follows:  The  immune  body 
(hsemolysin  heated  to56°  C.)  is  added  to  the  red  blood-cells  and,  after 
a  certain  time,  the  mixture  is  centrifuged.  The  clear  fluid  so  obtained 
is  tested  for  free  immune  body  by  adding  an  excess  of  complement 
and  allowing  this  mixture  to  act  on  the  same  quantity  of  fresh  blood- 
cells.  If  one  proceeds  in  this  manner  in  a  large  series  of  cases,  employ- 
ing varying  multiples  of  the  solvent  dose  of  immune  body,  it  is  possible 
to  determine  accurately  the  combining  power  of  the  cells.  The 
following  experiment  will  very  readily  make  this  clear. 

The  immune  body  was  present  in  the  serum  of  a  sheep  which 
had  been  treated  with  dog  blood.  When  this  serum  was  inactivated 


76  COLLECTED  STUDIES  IN  IMMUNITY 

by  heating  to  56°  C.,  it  could  be  reactivated  either  with  the  com- 
plement of  sheep  serum  or  of  goat  serum.  To  begin,  the  exact 
quantity  of  immune  body  was  determined  which  would  just  com- 
pletely dissolve  2  cc.  of  a  5%  mixture  of  dog  blood-cells  when  suf- 
ficient complement  was  present.  This  dose  was  found  to  be  0.15  cc. 
To  a  number  of  separate  portions  of  blood  mixture  (each  of  2  cc.)  mul- 
tiples of  this  dose  were  then  added,  thus,  1,  1J,  1^,  If,  2,  2J,  3  times 
the  solvent  dose,  and  the  mixtures  kept  at  room  temperature  for  an 
hour  and  frequently  shaken.  Since  the  complement  was  absent, 
haemolysis  could  not  occur.  After  centrifuging,  the  clear  fluid, 
which  had  the  appearance  of  water,  was  again  mixed  with  the  corre- 
sponding amount  of  blood  (0.1  cc.  of  undiluted  blood)  and  with 
complement.1  It  was  found  that  even  the  last  trace  of  the  single 
solvent  dose  had  disappeared  from  the  fluid;  whereas  in  the  case 
where  double  the  dose  had  been  added,  the  fluid  still  contained  just 
a  solvent  dose,  i.e.,  it  completely  dissolved  the  freshly  added  blood- 
cells.  In  this  case,  therefore,  the  blood-cells  were  able  to  combine 
with  only  a  single  dose  of  the  immune  body. 

This,  however,  is  not  at  all  the  general  rule,  for  by  extendiny 
our  experiments  to  other  cases  we  found  that  there  is  a  very  large 
variability  in  this  binding  of  the  immune  body,  and  that  frequently 
a  larger  or  smaller  multiple  of  the  solvent  dose  is  bound.  The  follow- 
ing case  will  illustrate  the  extreme  in  the  other  direction,  in  which 
almost  a  hundred  times  the  solvent  dose  of  immune  body  was  taken  up 
by  the  blood-cells.  A  rabbit  had  been  treated  with  goat  blood,  and  its 
serum  therefore  contained  an  immune  body  fitting  to  goat  blood.  Nor- 
mal guinea-pig  serum  served  as  complement  and  0.2  cc.  represented 
considerably  more  than  sufficient  for  2  cc.  of  the  goat  blood  mixture. 
When  this  amount  of  complement  was  employed,  the  solvent  dose 
of  the  immune  body  for  2  cc.  of  the  blood  mixture  amounted  to 
0.008  cc.  On  allowing  0.48  cc.  (sixty  times  the  solvent  dose)  to 
act  on  the  blood-cells  in  the  manner  previously  described,  and  then 
centrifuging,  it  was  found  that  the  clear  fluid  did  not  contain  even 
a  trace  of  immune  body.  When  eighty  times  the  dose  was  employed 
the  clear  fluid  showed  a  very  faint  solvent  action,  corresponding  to 
about  -J-  to  J  of  a  solvent  dose.  Not  until  one  hundred  times  the  dose 

1  As  a  counter  test  the  blood-cells  separated  by  centrifuge  were  mixed  with 
salt  solution  and  with  the  complement.  Those  specimens  in  which  just  the 
solvent  dose  (0.15  cc.)  of  the  immune  body  or  more  was  present,  dissolved. 
completely. 


STUDIES   OX  ILEMOLYSIXS.  77 

was  employed  did  the  centrifuged  fluid  contain  a  full  solvent  dose 
and  effect  complete  solution.  Hence  out  of  one  hundred  solvent 
doses  about  ninety-nine  had  been  bound  by  the  blood-cells,  for 
only  about  one  solvent  dose  of  immune  body  remained  in  the  fluid. 
By  means  of  parallel  experiments  we  have  found  that  one  hour's 
contact  of  immune  body  with  blood-cells  results  in  the  maximum 
amount  of  binding,  for  the  experiments  at  45°  C.  and  room  tem- 
perature yielded  results  exactly  alike.  Between  the  extremes  repre- 
sented by  these  two  experiments  a  great  variety  of  figures  was 
obtained. 

The  significance  of  these  experiments  offers  no  difficulties  from 
the  point  of  view  of  the  side-chain  theory.  The  facts  are  readily 
understood  when  we  stop  to  consider  the  peculiarities  of  the  receptor 
apparatus  of  the  blood-cells.  As  a  result  of  our  previous  experiments 
on  the  isolysins  of  goats  we  assume  that  a  given  blood-cell  contains  a 
large  number  of  different  types  of  receptors  which  in  general  fit  to 
different  immune  bodies  and  haemo toxins.  Referring  the  reader  to 
an  exhaustive  study  by  Ehrlich,1  we  shall  content  ourselves  here 
by  remarking  that  certain  kinds  of  receptors  may  be  present  in  the 
blood-cell  in  great  excess.  This  excess  cannot  only  be  demonstrated, 
but,  by  means  of  the  method  just  described,  can  be  exactly  measured. 
Entirely  analogous  conditions  arise  under  other  circumstances. 
The  interesting  fact  discovered  by  Wassermann,  that  the  central 
nervous  system  of  various  animals  binds  much  more  tetanus  poison 
in  vitro  than  is  necessary  to  fatally  poison  the  animal,  is  probably 
due  to  such  an  excess  of  receptors  for  tetanus  poison. 

From  this  point  of  view  the  experiments  above  mentioned  are 
easily  explained  without  departing  from  the  side-chain  theory.  Thus, 
let  us  assume  that  with  a  certain  poison  a  it  is  necessary  that  x  a-re- 
ceptors  are  bound  in  order  that  a  blood-cell  be  completely  dissolved, 
and  let  us  further  assume  that  the  blood-cell  posseses  a  much  greater 
number,  say  2x  a-receptor^.  When  Bordet's  experiment  is  now 
carried  out,  the  conditions  arising  will  be  exactly  those  described 
by  Bordet.  It  is  at  once  apparent  that  the  red  blood-cell  in  this 
case  will  combine  with  just  twice  the  amount  of  poison  necessary 
for  its  solution.  If  therefore  double  the  solvent  dose  of  immune 
body  is  added  to  a  given  amount  of  such  blood-cells,  the  entire  receptor 

1  Specielle  Pathologie  und  Therapie,  edited  by  Xothnagel,  Vol.  VIII,  sec- 
tion 3,  pages  163-184. 


78  COLLECTED   STUDIES   IN  IMMUNITY. 

system  of  these  cells  will  be  occupied.  On  adding  now  an  equal 
portion  of  fresh  blood,  the  latter  will  fail  to  find  any  free  immune 
body  and  cannot  therefore  be  attacked. 

Such  phenomena  are  exceedingly  plentiful  in  chemistry,  and  it 
may  pay  us  to  glance  at  some  of  them.  Napthalin,  as  is  well  known, 
consists  of  two  benzole  nuclei  joined  together.  When,  now,  a  salt- 
forming  group,  hydroxyl  or  amido  group,  is  introduced  into  each  of 
the  two  benzole  nuclei,  the  heteronuclear  substitution  products,  e.g., 
dioxynaphthalin,  amidonaphthol,  and  naphthylenediamine,  or  their 
sulfo  acids,  will  be  able  to  combine  with  either  one  or  with  two  mole- 
cules of  a  diazo  combination.  When  two  molecules  of  dioxynaph- 
thalin are  mixed  with  two  molecules  of  diazobenzol,  the  result  is  ex- 
clusively the  mono-azo  combination;  when  however  two  molecules 
of  diazobenzol  are  added  to  one  molecule  of  dioxynaphthalin,  the  result 
is  the  diazo  combination.  If  an  additional  molecule  of  dioxynaph- 
thalin is  added  to  the  finished  diazo  combination,  this  molecule  will 
be  unable  to  dissociate  the  latter,  and  the  two  substances,  the  diazo 
combination  and  the  unchanged  dioxynaphthalin,  will  exist  side  by 
side.  This  example,  to  which  others,  such  as  the  esterification  of 
dibasic  acids,  the  methylation  of  anilin  with  iodomethyl,  could 
easily  be  added,  corresponds  entirely  to  the  relations  between  im- 
mune body  and  erythrocytes  as  described  by  Bordet. 

It  may  at  once  be  admitted  that  where  the  binding  of  small 
multiples  of  the  immune  body  is  concerned,  it  is  very  natural  to 
think  of  a  mechanical  absorption  due  to  the  degree  of  concentration  ; 
and  that  therefore  the  circumstances  in  Bordet 's  case,  in  which  the 
binding  was  merely  doubled,  justified  the  comparison  with  staining 
processes.  The  cases  examined  by  us,  however,  in  which  at  one  time 
just  the  solvent  dose  of  immune  body,  at  another  an  extraordinarily 
large  multiple  of  the  dose  was  bound,  weigh  heavily  against  this 
assumption. 

Our  decision,  however,  is  especially  determined  by  certain  general 
considerations.  Thus,  charcoal,  the  type  of  surface-attractive  agenis 
attracts  thousands  of  substances  of  the  most  varied  kind.  A  dye  can 
stain  a  large  number  of  different  substances,  as  is  shown  in  every 
stained  microscopic  preparation.  In  marked  contrast  to  this  is 
the  specificity  of  the  numerous  antibodies,  which  primarily  are 
always  directed  against  the  exciting  bacterial  or  other  cell 
species. 

In  the  cases  in  which  apparent  deviations  from  this  rule  were. 


STUDIES  ON   ELEMOLYSINS.  79 

noted,  exact  investigation  has  shown  1  that  these  are  due  to  the  pres- 
ence of  one  and  the  same  receptor  group  in  various  elements.  Thus 
we  have  shown  that  the  isolysins  produced  by  injecting  goats  with 
goat  blood-cells  act  also  on  sheep  blood-cells.  We  have  further 
shown  that  these  sheep  blood-cells  possess  certain  lands  of  receptors 
which  bind  the  goat  lysin  just  as  the  receptors  which  are  present  in 
the  goat  blood-cells  do.  We  produced  the  strongest  proof  for  this 
community  of  receptors  by  means  of  crossed  immunization,  for  we 
succeeded  in  producing  a  typical  isolysin  by  injecting  goats  with 
sheep  blood. 

Since  all  experiences,  therefore,  lead  us  to  assume  that  each  par- 
ticular complex  produces  just  the  specific  antibody,  and  since  this 
agrees  exceedingly  well  with  the  assumption  of  a  chemical  union, 
it  would  be  a  distinct  backward  step  to  adopt  so  vague  a  conception 
as  that  of  mechanical  surface  attraction.  . 

Were  we  to  assume  that  the  immune  body  enters  the  cell  merely 
mechanically,  it  would  be  necessary  to  drop  the  entire  unity  of  the 
immunization  phenomena  which  follows  from  the  side-chain  theory. 
It  is  probably  quite  generally  conceded  that  the  antitoxin  acts  on 
the  toxin  in  a  purely  chemical  manner.  Hence  so  far  as  dissolved 
substances  developed  by  the  immunity  reaction  are  concerned,  the 
chemical  conception  applies.  Why  then  should  this  chemical  action 
suddenly  cease  when  the  substances  instead  of  being  in  solution  are 
present  within  the  cell,  and  a  new  principle  be  assumed  for  this 
case?  This  leads  to  the  contradiction  that  in  one  case  (when  com- 
bining with  the  erythrocytes)  the  immune  body  is  bound,  specifically 
to  be  sure,  but  mechanically,  while  in  the  othfer  case  (when  anchored 
to  an  artificially  produced  anti-immune  body  in  solution)  it  is  bound 
specifically  but  chemically. 

These  considerations,  and  they  could  readily  be  greatly  extended, 
will  suffice  to  show  that  the  above-mentioned  experiments  are  not 
at  all  capable  of  shaking  the  side-chain  theory,  for  by  it  alone  is 
a  single  uniform  conception  of  the  phenomena  of  immunity  rendered 
possible. 

II.  Concerning  Complementoids. 

The  complements,  which  effect  the  activation  of  the  normal 
immune  bodies  and  of  those  produced  by  immunization  (amboceptors) 
do  not  possess  great  theoretical  or  practical  importance  in  the  study 

1  See  Third  Communication,  page  23. 


80  COLLECTED  STUDIES  IN  IMMUNITY. 

of  immunity.  They  seem  to  play  an  important  role  in  the  normal 
processes  of  cell  nutrition.  As  a  result  of  experiments  already 
described  we  must  assume  that  in  the  blood  serum  of  a  particular 
animal  species  not  merely  a  single  complement  exists  but  a  large 
number  of  different  complements.  It  is  understood,  of  course,  that 
not  all  the  complements  occurring  in  a  large  number  of  species  differ 
from  one  another.  On  the  contrary  it  is  to  be  regarded  as  certain 
that  particular  types  find  a  wide  distribution  extending  over  several 
animal  species.  This  explains  why,  for  example,  a  hsemolytic  or 
bacteriolytic  immune  body  can  be  reactivated  by  the  sera  of 
different  animal  species. 

We  have  previously  explained  that  a  complement  is  to  be  con- 
ceived as  possessing  two  characteristic  groups,  a  haptophore  group 
which  fits  into  the  complementophile  group  of  the  immune  body, 
and  a  zymotoxic  group  which  is  the  actual  carrier  of  the  specific 
action.  A  complement  therefore,  to  a  certain  extent,  corresponds  to 
a  toxin,  which  possesses  a  haptophore  and  a  toxophore  group.  Hence 
by  the  immunization  of  suitable  animals  it  is  easy  to  obtain  anti- 
complements  whose  behavior  corresponds  exactly  to  that  of  anti- 
toxins. For  example,  if  a  goat  or  rabbit  is  injected  with  horse 
serum,  an  anticomplement  will  be  formed  which  is  able  to  specifically 
inhibit  the  action  of  the  complement  contained  in  horse  serum.  We 
have  already  shown  1  that  this  is  due  to  a  deflection  of  the  comple- 
ment. 

We  have  now  tried  to  follow  this  analogy  (between  complements 
and  toxins)  further.  We  take  it  for  granted  that  it  is  generally 
known  that  toxins,  either  through  spontaneous  changes  or  through 
the  action  of  chemical  agents,  become  modified  into  toxoids,  whose 
distinguishing  character  is  that  they  no  longer  possess  a  toxophore 
group  although  the  haptophore  group  remains.  These  toxoids,  then, 
are  relatively  non-toxic  substances  which  are  nevertheless  able  to 
cause  the  formation  of  antitoxins  in  the  animal  body.  Now  we 
know  that  the  zymotoxic  group  is  extremely  sensitive  to  the  most 
varied  influences;  hence  the  attempt  to  study  modifications  of  the 
complements  analogous  to  the  toxoids  seemed  to  promise  favorable 
results.  Such  modified  complements  would  then  be  designated 
complementoids.  The  first  step  was  to  see  whether  the  well-known 
inactivation  of  a  serum  by  heating  to  56°  C.  completely  destroyed 

— 
1  See  Fourth  Communication,  page  56. 


STUDIES  OX  ELEMOLYSINS.  81 

the  complements  or  merely  changed  them  into  inactive  derivatives, 
complementoids.1: 

In  order  to  be  certain  of  the  destruction  of  the  zymotoxic  group, 
we  heated  the  sera  for  fifty  minutes  to  60°  C.,  a  procedure,  as  shown 
by  numerous  subsequent  examinations,  which  absolutely  destroys 
every  trace  of  complement  action  in  the  sera  so  treated. 

By  treating  animals  with  the  sera  thus  prepared,  it  is  actually 
very  easy  to  obtain  anticomplements.  We  injected  rabbits,  guinea- 
pigs,  and  dogs  with  inactive  goat  serum,  and  goats  and  numerous 
rabbits  with  inactive  horse  serum.  A  parallel  series  of  animals 
was  treated  with  active  serum.  The  anticomplement  action  of 
the  serum  from  the  animals  treated  with  complementoids  proved 
fully  as  strong  and  often  stronger  than  that  of  the  control  animals 
treated  with  active  serum.  By  means  of  the  procedure  described 
in  detail  in  our  Fourth  Communication  it  was  readily  shown  that 
these  were  really  anticomplements. 

The  injection  of  the  heated  serum,  therefore,  possesses  the  same 
value  as  that  of  the  unchanged  serum.2  Since,  however,  accord- 
ing to  our  view  it  is  the  haptophore  group  which  causes  the  immu- 
nity reaction,  it  follows  that  inactivation  of  the  complement  has  de- 
stroyed only  the  zymotoxic  group,  leaving  the  haptophore  group  intact. 

The  important  question  now  arises  as  to  how  the  presence  of 
complementoids  influences  the  activation  of  the  immune  body; 
for  whenever  a  serum  is  inactivated  by  heating  a  formation  of  com- 
plementoid  ensues,  and  it  is  well  known  that  such  a  mixture  of  immune 
body  and  complement  is  reactivated  without  any  trouble  by  the 
addition  of  complement.  It  seems  therefore  as  though  the  presence 
of  the  complementoid  does  not  hinder  the  union  of  immune  body 
and  complement. 

On  this  point  we  have  made  special  experiments  by  alternately 

1  At  about  the  same  time,  exactly  similar  considerations  led  Paul  Miiller 
(Centralblatt  f.    Bacteriologie,  Vol.   29,  No.   5)   to  attempt   the  production  of 
anticomplement  by  the  injection  of  serum  which  had  been  heated.     In  his 
case,  however  (immunization  with   chicken    blood),  anti-interbody  was    prin- 
cipally  developed,    while    anticomplement    could   not    positively  be    demon- 
strated.    It  is  possible  that  this  negative  result  indicates  that  not  all  the  com- 
plements of  the  different  animal  species  are  able  to  undergo  this  metamorphosis 
into  complementoid. 

2  We  should  like  to  mention  that  in  addition  to  this,  in  the  case  of  the  goat 
treated  with  inactive  horse  serum,  we  observed  the  development  of  a  powerful 
coagulin. 


82  COLLECTED  STUDIES  IN  IMMUNITY. 

inactivating  and  adding  complement  without  finding  that  the  con- 
stantly increasing  amount  of  complementoid  hindered  the  action 
of  the  complement.  This  phenomenon  can  be  explained  only  by 
assuming  that  in  the  change  to  complementoid ,  the  haptophore  group 
of  the  complement  suffers  a  diminution  of  its  affinity  for  the  comple- 
mentophile  group  of  the  immune  body. 

In  the  toxoids  of  diphtheria  poisons  the  circumstances  are  some- 
what different,  for  Ehrlich  found  that  in  the  hemitoxin  zone  of  the 
poison  spectrum  the  affinity  suffers  no  change  through  the  forma- 
tion of  toxoid.  On  the  other  hand,  M.  Neisser  and  Wechsberg  in 
another  case,  namely  that  of  staphylotoxin,  have  been  able  to  demon- 
strate a  decrease  in  affinity  occurring  with  the  change  into  toxoid. 
This  behavior  is  analogous  to  that  of  the  complementoids  observed 
by  us.  Hence  no  general  rules  governing  the  affinities  in  toxoid 
and  complementoid  formation  can  be  laid  down;  the  circumstances 
must  be  investigated  separately  in  each  case.  From  what  slight 
differences  in  the  constitution  of  the  molecule  enormous  differences 
in  affinity  may  arise  is  seen  by  studying  certain  organic  acids.  Thus, 
for  example,  a  and  /?  resorcylic  acids  differ  from  each  other  merely  in 
the  position  of  the  two  hydroxyl  groups;  the  constants  of  their 
affinities,  however,  differ  from  each  other  by  over  a  hundred  times. 
We  may  therefore  perhaps  assume  that  in  our  special  case  it  depends 
on  the  relative  positions  of  the  haptophore  and  hoxophore  group 
and  the  corresponding  relations  thereby  determined  whether  any 
change  in  one  group  can  retroactively  affect  the  other. 

III.    Concerning  Autoanticomplements. 

In  the  third  communication,  on  isolysins,  we  pointed  out  that 
the  organism  possesses  certain  contrivances  by  means  of  which 
the  immunity  reaction,  so  easily  produced  by  all  kinds  of  cells,  is 
prevented  from  acting  against  the  organism's  own  elements  and 
so  give  rise  to  autotoxins.  Further  investigations  made  by  us  have 
confirmed  this  view,  so  that  one  might  be  justified  in  speaking  of 
a  "horror  autotoxicus"  of  the  organism.  These  contrivances  are 
naturally  of  the  highest  importance  for  the  existence  of  the  indi- 
vidual. During  the  individual's  life,  even  under  physiological  though 
especially  under  pathological  conditions,  the  absorption  of  all  material 
of  its  own  body  can  and  must  occur  very  frequently.  The  formation 
of  tissue  autotoxins  would  therefore  constitute  a  danger  threatening 
the  organism  more  frequently  and  much  more  severely  than  all 


STUDIES  ON  H^VIOLYSINS.  83 

exogenous  injuries.  We  believe  that  the  study  of  these  regulating 
contrivances  is  of  the  greatest  importance  and  according  to  our 
present  investigations  either  the  disappearance  of  receptors  or  the 
presence  of  autoantitoxins  is  foremost  among  these  contrivances. 
It  will  therefore  be  necessary  to  subject  all  the  factors  which  are  of 
importance  in  this  respect  to  a  thorough  analysis.1 

We  shall  now  mention  a  few  observations  relating  to  the  com- 
plements which  seem  to  point  to  a  regulatory  contrivance  as  yet 
undescribed. 

Normal  rabbit  serum  possesses  a  number  of  properties  which 
are  to  be  ascribed  to  the  presence  of  complements.  First  to 
be  mentioned  is  the  property  by  means  of  which  freshly  derived 
rabbit  serum  is  able  to  dissolve  guinea-pig  blood-cells.  This  is  due 
to  the  combined  action  of  a  complement  and  an  immune  body  which 
is  present  in  the  serum  in  comparatively  small  amounts.  Further- 
more, rabbit  serum  is  regularly  able  to  activate  an  immune  body 
derived  by  treating  rabbits  with  ox  blood. 

Now  we  noticed  that  rabbits  which  a  week  previously  had  been 
treated  with  goat  serum  (whether  active  or  inactive  is  immaterial) 
had  completely  or  almost  completely  lost  these  properties,  and  that 
these  changes  persisted  for  weeks  after  the  injection.  Hence  it  fol- 
lows that  owing  to  the  injection  of  goat  serum,  complement  nor- 
mally present  had  been  made  to  disappear.  It  was  therefore  essen- 
tial that  the  cause  of  this  remarkable  phenomenon  be  determined. 
We  could  next  show  that  frequently  the  serum  of  these  rabbits  in 
its  native  state,  though  more  surely  after  heating  to  56°  C.,  is  able 
to  prevent  the  above-described  complementary  action  of  normal 
rabbit  serum.  Hence  in  the  above  case  normal  complement  has 
evidently  disappeared  from  the  rabbit  treated  as  described,  and 
has  been  replaced  by  an  anticomplement  which  we  shall  have  to 
term  an  a utoant {complement.2 

1  Metalnikoff's  interesting  observation    is  only  apparently  a  contradiction 
of  these  regulating  phenomena.     He  found  that  a  typical    autospermotoxin  is 
developed  in  the  blood  of  guinea-pigs  which  have  been  treated  with  guinea-pig 
spermatozoa,  and  that  this  is  able  in  vitro  to  kill  the  spermatozoa  of  the   animal 
itself.     But  such  an  injurious  action  on  the  spermatozoa  does  not  take  place, 
even  in  the  slightest  degree,  in  the  living  animal,  because,  as  Metalnikoff's 
researches  show,  only  the  immune  body  combines  with  the  spermatozoa,  not 
the  complement.     In    this  case,  therefore,  an  autotoxin  within  our  meaning, 
one  that  destroys  the  cells  of  its  own  body,  does  not  exist. 

2  According  to  the  investigations  of  Dr.  M.  Xeisser  and  Dr.  Wechsberg  still 


84  COLLECTED  STUDIES  IN  IMMUNITY. 

It  has  previously  been  shown  that  such  a  rabbit  serum  is  rich  in 
antigoat  complement.  We  observed  an  analogous  phenomenon, 
whose  nature  may  perhaps  be  identical  with  the  above,  in  a  rabbit 
which  had  been  treated  with  ox  blood  (blood-cells  and  serum)  in 
order  to  produce  a  specific  hsemolysin.  Ten  days  after  the  injection 
of  ox  blood,  the  serum  failed  to  show  any  solvent  action  whatever 
on  ox  blood,  in  direct  contrast  to  numerous  previous  cases.  At 
first  we  thought  it  possible  that  no  immune  body  had  been  formed 
in  this  case,  for  even  the  addition  of  an  excess  of  complement 
in  the  form  of  rabbit  serum  produced  no  solution.  However,  on 
<?entrifuging  the  ox  blood-cells  after  treatment  with  this  abnormal 
serum,  and  mixing  them  with  salt  solution  and  complement,  we 
found  that  even  slight  doses  of  immune  serum  caused  marked  so- 
lution. The  serum  therefore  contained  plenty  of  immune  body, 
and  this  had  been  anchored  by  the  blood-cells.  The  presence  of 
this  immune  body  was  obscured  not  only  because  the  complement 
was  absent,  but  because  this  had  been  replaced  by  an  anticom- 
plement  which  neutralized  the  complement  subsequently  added. 
Because  of  the  anticomplement  which  it  contained,  this  rabbit 
serum  manifested  a  marked  inhibitory  action  on  the  strongly  hsem- 
olytic  serum  of  another  rabbit  (one  which  had  been  treated  with 
ox  blood). 

But  what  happened  in  this  case  after  injection  of  ox  blood  rarely 
occurs  in  such  a  conspicuous  manner.  More  frequently  it  is  found 
that  the  serum  in  its  active  state  possesses  an  exceedingly  slight 
solvent  action,  corresponding  to  a  very  small  content  of  complement, 
and  that  after  heating  it  manifests  a  distinct  anticomplementary 
action.  This  evidently  leads  to  the  extreme  case  above  described,  as 
is  readily  seen  when  the  relations  are  expressed  by  means  of  a  diagram. 
(See  figure.) 

In  studying  the  question  as  to  how  these  autoanticomplements 
are  formed,  we  must  constantly  bear  in  mind  that  normal  serum 
always  contains  complements  in  excess.  Now  it  is  difficult  to  see 
what  purpose  would  be  served  if  at  any  time  the  normal  comple- 
ments, so  important  in  cell  economy,  were  paralyzed  by  autoanticom- 
plements. We  shall  therefore  have  to  assume  that  the  normal 

in  progress,  this  serum  also  lacks  the  power  to  activate  certain  bactericidal 
immune  bodies.  The  animals  at  the  same  time  seem  to  suffer  a  decrease  in 
their  resisting  power  against  certain  infections,  a  fact  which  may  perhaps  serve 
to  exhibit  in  the  purest  form  the  function  of  certain  complements. 


STUDIES  ON  H^EMOLYSINS 


85 


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86  COLLECTED  STUDIES  IN  IMMUNITY. 

complements  circulating  in  the  serum  do  not  cause  the  formation  of 
autoanticomplements.  Confirmation  of  this  view  is  furnished  by 
the  fact  that  even  in  animal  species  possessing  identical  complements 
it  is  impossible  to  produce  anticomplements  by  means  of  serum 
injections.  Thus,  neither  sheep  when  injected  with  goat  serum,  nor, 
conversely,  goats  when  injected  with  sheep  serum  produce  any  anti- 
complement,  for  these  two  species  manifest  an  extensive  similarity 
in  their  complements  as  well  as  in  other  serum  constituents. 

When,  then,  in  spite  of  this  rule,  we  find  that  in  our  case  auto- 
anticomplements have  developed,  only  one  explanation  remains: 
that  one  or  the  other  complement  present  in  the  goat  serum,  although 
related,  is  not  identical  with  the  complement  of  the  rabbit.  If  we  assume 
that  a  certain  goat  complement  possesses  the  same  haptophore  group 
as  does  a  certain  rabbit  complement,  but  that  it  differs  in  the  rest  of 
its  constitution,  then  the  assumption  that  identical  complements  do  not 
form  anticomplements  will  not  apply.  In  this  case,  by  means 
of  the  haptophore  group  of  the  particular  receptor  of  the  rabbit  cell, 
a  foreign  complex  would  be  anchored  which  exerts  a  sufficient  stimulus 
on  the  cell  to  cause  an  increased  production  and  thrusting  off  of  the 
corresponding  side-chains  which  can  functionate  as  anticomplements. 

We  shall  have  to  assume  that  the  particular  goat  complement, 
because  of  its  identical  haptophore  group,  can  be  anchored  at  the 
same  places  as  the  idiocomplements  with  the  same  haptophore  group. 
Foremost  among  these  places  we  may  consider  the  complex  receptors 
which  possess  two  haptophore  groups  (amboceptors).  In  this  case, 
contrary  to  what  we  usually  observe,  the  thrusting-off  of  an  amboceptor 
would  be  effected  through  the  anchoring  of  its  complementophile  group, 
and  we  should  then  have  additional  proof  for  our  view  that  the  com- 
plex receptors  possess  two  binding  groups. 

In  any  case  it  would  seem  to  be  of  the  greatest  importance  to  gain 
an  insight  into  the  conditions  governing  the  disappearance  of  the 
idiocomplements.  That  they  can  be  caused  to  disappear  through 
injection  of  anticomplements  produced  by  immunization  follows  as 
a  matter  of  course  from  our  definition  of  anticomplements.  This, 
however,  occurs  only  under  artificial  experimental  conditions  and 
so  possesses  but  little  significance  pathologically.  Of  considerable 
importance  for  these  occurrences  under  natural  circumstances  are 
the  vital  conditions  governing  the  disappearance  of  complement 
through  internal  metabolic  processes.  The  origin  of  the  autoanti- 
complements as  it  has  just  been  presented  by  us  surely  belongs  here, 


STUDIES  ON  IL^MOLYSINS.  87 

and  it  has  perhaps  some  practical  significance,  viz.,  that  in  the  fre- 
quent injection  of  various  curative  sera  into  man  and  animals,  the 
possibility  of  autoanticomplement  formation  should  be  borne  in 
mind.  Another  case  belonging  here  has  previously  been  described 
by  us — the  disappearance  of  part  of  the  complements  in  a  rabbit 
poisoned  with  phosphorus.  In  connection  with  this  the  following 
observation  of  Metalnikoff  (1.  c.)  is  of  interest.  He  was  immunizing 
a  rabbit  with  spermatozoa  and  noticed  that  in  consequences  of  a 
purulent  process  which  developed  during  the  course  of  the  immuni- 
zation, the  complement  which  activated  the  spermotoxin  disappeared 
from  the  serum  and  did  not  reappear  for  a  considerable  time. 

These  isolated  observations  seem  to  indicate  that  the  com- 
plements can  disappear  during  pathological  conditions  in  conse- 
quence, perhaps,  of  a  more  rapid  destruction  or  of  a  slower  formation. 
The  same  holds  true  for  the  immune  bodies  (amboceptors)  which 
in  bacteriolysis  as  well  as  in  haemolysis  have  at  least  as  great  a  sig- 
nificance as  the  complements.  Which  of  these  two  factors  prevails 
in  any  single  case  cannot  be  decided  by  any  general  rule,  but  each  case 
must  be  examined  separately.  Only  through  such  investigation  will 
we  gain  an  insight  into  the  nature  of  "natural  predisposition"  and 
its  changes,  " increased  resistance/'  "loss  of  resistance,"  etc. 


VEIL    STUDIES  ON  ILEMOLYSINS.1 

SIXTH  COMMUNICATION. 
By  Prof.  Dr.  P.  EHBLICH  and  Dr.  J.  MORGENROTH. 

THE  steady  progress  of  the  investigations  in  immunity  is  rendered 
extremely  difficult  by  the  fact  that  in  the  immunization  with  living 
cells  and  in  the  study  of  the  immune  sera  thus  obtained  a  large  number 
of  different  substances  which  exist  simultaneously  is  concerned. 
In  our  second  communication  we  pointed  out  that  the  hsemolysins 
present  in  normal  serum,  which  act  on  different  species  of  blood,  are 
not  a  single  substance  in  the  sense  of  Buchner's  alexin;  and  in  our 
fourth  communication  we  showed  that  this  could  be  demonstrated 
experimentally  by  means  of  elective  absorption.  It  is  possible  that 
just  as  many  interbodies  come  into  action  as  the  varieties  of  blood 
affected.  We  have  also  been  unable  to  accept  Bordet's  Unitarian 
view  of  the  complements.  On  the  contrary,  as  a  result  of  our  own 
experiments  we  have  become  convinced  that  a  large  number  of  com- 
plements exist  together  in  blood  serum.  In  like  manner  Bordet's 
absorption  experiments  indicate  a  plurality  of  the  bacterial  agglutinins 
and  those  of  Malkoff  a  plurality  of  the  normal  hsemagglutinms. 
The  results  of  these  experiments  have  been  gathered  together  by  M. 
Neisser  2  in  a  study  in  which,  on  the  basis  of  the  same  principles,  he 
demonstrates  the  variety  of  the  antitoxic  antibodies  occurring  in  nor- 
mal serum.  In  conformity  to  this,  the  reactive  antibodies  produced  by 
injections  of  serum  of  foreign  species  are  most  varied  in  their  nature, 
and  we  are  only  just  beginning  to  gain  an  insight  into  their  constitution. 
Aside  from  the  numerous  coagulins  and  antiferments  thus  produced, 
it  is  of  the  utmost  importance,  so  far  as  the  discussion  of  immunity 
problems  is  concerned,  to  recognize  the  fact  that  the  complements 

1  Reprinted  from  the  Berliner  klin.  Wochenschr.,  1901,  Nos.  21  and  22. 

2  Deutsche  med.  Wochenschr.,  1900,  No.  49. 

88 


STUDIES  ON  H^MOLYSINS.  89 

formed  through  immunization,   corresponding  to  the  multiplicity  of 
the  complements  present  in  the  serum,  are  exceedingly  manifold. 

Especially  significant,  however,  is  the  fact  that  the  cells  possess 
a  great  number  of  different  kinds  of  groups,  which  groups  can  lead 
to  the  production  of  numerous  different  amboceptors  (immune 
bodies).1 

Hence  in  immunzing  an  animal  with  cell  material,  the  organism 
is  injected,  not  with  a  single  uniform  substance,  but  with  a  multitude 
of  the  most  varied  receptors,  each  of  which  is  more  or  less  able  to 
produce  an  antibody.  In  our  fourth  communication  we  defined  our 
point  of  view  on  this  basis  as  follows: 

"In  view  of  our  experiments  on  isolysins  described  in  our  third 
communication  the  occurrence  of  different  immune  bodies  in  a 
haemolytic  serum  obtained  by  immunizing  with  red  blood-cells  is 
not  at  all  surprising.  We  have  obtained  a  whole  series  of  different 
isolysins  by  injecting  goats  with  goat-blood.  At  present  they  number 
twelve.  In  the  red  blood-cells  not  merely  a  single  group,  but  a  large 
number  of  different  groups,  must  be  considered,  which,  provided 
there  are  fitting  receptors,  can  produce  a  corresponding  series  of 
immune  bodies.  All  of  these  immune  bodies,  again,  will  be  anchored 
by  the  blood-cells  employed  in  immunization.  "We  may  assume 
that  when  an  animal,  species  A,  is  immunized  with  blood-cells  of 
species  B,  a  hamolytic  serum  will  be  produced  which  contains  a  great 
host  of  immune  bodies.  The  immune  bodies  in  their  entirety  are 
anchored  by  the  blood-cells  of  species  A." 

Durham2  has  adopted  the  same  view  for  the  bacterioagglutinins. 
He  assumes  a  number  of  "  components  "  (corresponding  to  our  recep- 
tors) in  the  body  substance  of  the  bacteria,  which  can  cause  the 
production  of  a  corresponding  number  of  agglutinins.  In  this  way 
each  agglutinin  which  acts  on  a  certain  species  of  bacteria  represents 
the  sum  of  different  kinds  of  single  agglutinins,  a  view  entirely 
analogous  to  our  assumption  of  a  plurality  of  immune  bodies.  This 
view  permits  Durham  to  offer  a  sufficient  and  natural  explanation 
of  the  varying  degree  of  action  of  typhoid  agglutinins  on  typhiod 
bacilli  of  different  origin,  and  of  the  extension  of  the  agglutinating 
action  of  specific  sera  to  related  species  of  bacteria.  It  would  be 


1  Compare  the  thorough  exposition  by  Ehrlich  in  Vol.  VIII  of  NothnagePs 
Specielle  Pathologic  imd  Therapie,  Holder,  Vienna,  1901. 

1  Durham,  Journ.  of  Experimental  Medicine,  Xew  York,  Vol.  V,  No.  4,  1901. 


SO  COLLECTED  STUDIES  IN  IMMUNITY. 

Tery  interesting  to  see  these  as  yet  purely  theoretical  suggestions 
of  Durham  proved  by  means  of  experiments. 

The  pluralistic  standpoint  adopted  by  us  creates  numerous 
difficulties  for  thorough  analytical  work  in  this  field,  but  it  leads  to  a 
deeper  insight  into  the  complicated  problems  and  may  perhaps 
also  prove  of  value  in  the  practical  applications  in  immunity. 

I.  Observations  on  the  Pluralistic  Conception  of  the  Cellular 
Immunity  Reaction. 

To  begin,  we  shall  briefly  sketch  one  of  the  points  of  view  yielded 
by  the  plurimistic  conception,  which  seems  to  be  of  some  practical 
value.  Let  us  assume  that  a  cell,  e.g.,  a  bacterial  cell,  possesses 
twenty  different  groups ;  then  twenty  different  antibodies  correspond- 
ing to  these  will  be  possible.  Each  haptophore  group  of  the  bacterial 
cell  will  then  represent  an  isolated  point  of  attack  for  one  particular 
immune  body.  It  is  certainly  most  logical  to  conclude  that  the 
possibility  of  successfully  combating  a  certain  bacterial  infection 
increases  directly  with  the  number  of  kinds  of  immune  bodies  which 
act  on  the  bacterial  cell.1  The  ideal  effect  would  obviously  be  attained 
if  it  were  possible  to  produce  a  serum  so  constituted  as  to  contain 
immune  bodies  for  all  the  groups  present  in  the  bacterial  cell. 

The  phenomenon  of  antibody  formation  as  it  proceeds  according 
to  the  side-chain  theory  is  a  very  complex  one,  and  is  composed 
•of  a  number  of  phases  (binding,  super-regeneration,  thrusting-off) 
which  are  partly  independent  of  each  other.  Hence  a  variety  of 
circumstances  may  arise  which  exert  an  inhibitory  action  at  certain 
points. 

To  begin,  the  cell  may  be  so  severely  damaged  by  the  anchoring 
of  certain  poisonous  substances  that  the  formation  of  antibody 
does  not  occur  at  all,  or  occurs  in  only  a  very  slight  degree ;  for  this 
antibody  production,  which  is  a  kind  of  regeneration  process,  pre- 
supposes a  certain  degree  of  cell  efficiency.2 

This  damaging  effect  will  result  especially  with  highly  toxic  sub- 

1  It  is,  in  fact,  conceivable  that  the  occupation  of  a  single  group  only  produces 
a  certain  amount  of  injury  to  the  cell  without  being  able  to  cause  its  death.     The 
danger  to  the  life  of  the  bacterial  cells  would  increase  in  proportion  to  the  number 
of  partial  injuries,  which  again  would  correspond  to  the  increase  in  the  number 
of  types  of  receptors.     It  is  possible  that  the  potent  bactericidal  sera  so  far 
obtained  owe  their  success  to  a  certain  plurality  of  the  immune  bodies. 

2  Weigert    has   already  called  attention  to  this.     See    Lubarsch-Ostertag, 
Ergebnisse  der  Pathologic,  1897,  page  138. 


STUDIES  OX   ILEMOLYSIXS.  91 

stances,  provided  the  receptors  fitting  these  are  present  exclusively 
in  vitally  important  organs,  e.g.,  the  central  nervous  system.  This 
perhaps  explains  the  circumstance  that  it  is  exceedingly  difficult 
to  produce  an  antitoxin  in  mice  and  guinea-pigs  with  unchanged 
tetanus  poison,  while  this  is  easily  effected  when  toxoids  are  used. 
On  the  other  hand,  an  immunization  of  rabbits  by  means  of  unchanged 
tetanus  poison  is  very  easy  to  attain,  because  in  these  animals, 
as  is  shown  by  the  investigations  of  Donitz  and  of  Roux,  the  greater 
part  of  the  receptors  lies  outside'  of  the  poison-endangered  central 
nervous  system. 

However,  even  without  any  development  of  illness  it  is  not  at 
all  necessary  that  antibodies  should  be  produced  in  every  case  hi 
which  an  anchoring  occurs.  Metchnikoff,  for  example,  has  called 
attention  to  the  fact  that  with  frogs  in  whom  every  trace  of  illness 
is  avoided  by  keeping  them  cool  (as  we  know  from  Courmont's  beau- 
tiful observations)  it  is  impossible  to  produce  any  tetanus  antitoxin. 
Investigations  by  Morgenroth  have  confirmed  this  result  and  shown 
further  that  even  by  treatment  with  toxoids  under  various  conditions 
it  is  impossible  to  produce  a  trace  of  immunity.  Probably  in  this 
particular  case  these  results  indicate  that  the  regenerative  powers 
of  the  frog's  tissues  are  not  equal  to  these  extraordinary  demands. 

Such  an  explanation  for  failure  of  the  antibody  to 'develop  is,  how- 
ever, much  less  probable  in  the  case  of  warm-blooded  animals;  and 
as  the  number  of  experiments  increases,  these  cases  are  becoming 
more  frequent.  Probably  all  who  have  busied  themselves  with  the 
subject  will  have  found,  particularly  with  the  artificially  produced 
cell  poisons,  that  in  some  cases  it  is  extremely  difficult  if  not  impos- 
sible to  effect  the  production  of  anti-immune  bodies.  Thus, 
Metchnikoff  injected  a  series  of  guinea-pigs  with  specific  spermo- 
toxin,  a  substance  which  certainly  finds  receptors  hi  the  guinea-pig's 
organism.  Despite  this,  he  found  but  two  cases  in  which  even  a 
suggestion  of  antispermotoxin  could  be  demonstrated.  In  the 
case  of  a  dog  injected  with  a  specific  dog  blood  immune  body  derived 
from  a  sheep,  we  have  failed  despite  long-continued  treatment  to 
produce  any  anti-immune  body.  With  this  series  of  phenomena 
must  also  be  classed  the  fact  that  it  is  extremely  difficult  if  not  impos- 
sible in  a  number  of  animal  species  to  produce  antienzymes  by  the 
continued  injection  of  certain  enzymes. 

The  explanation  of  these  facts  presents  two  possibilities:  First, 
the  receptors  concerned  in  the  particular  case  may  be  of  peculiar 


92  COLLECTED  STUDIES  IN  IMMUNITY. 

constitution  in  one  respect,  i.e.  in  being  firmly  b'ound  to  the  proto- 
plasm, so  that  a  thrusting-off,  which  is  essential  for  the  formation 
of  antibodies,  does  not  occur  even  with  an  increased  regeneration 
(sessile  receptors).  This  leads  to  the  conception  that  the  regenera- 
tion of  the  receptors  may  take  two  courses:  (a)  a  thrusting-off 
of  the  receptors  ensues,  and  with  this  a  formation  of  antibody; 
(6)  in  the  case  of  sessile  receptors,  a  hypertrophic  process  sets  in 
comparable  perhaps  to  a  simple  muscle  hypertrophy,  according  to 
Weigert's  conception.  Second,  it  is  conceivable,  as  Morgenroth l 
has  shown  in  the  immunization  against  rennin,  that  normal  pre- 
formed regulating  contrivances  come  into  action,  for,  in  the  case  of 
enzymes  (in  contrast  to  toxins)  we  are  dealing  with  substances  nor- 
mally produced  by  the  organism  itself.  Hence  it  is  possible  that  the 
formation  of  an  antienzyme  is  followed  by  the  production  of  the 
enzyme  itself,  in  consequence  of  an  internal  regulating  contrivance. 

In  any  event  these  observations  will  show  how  the  factors  just 
discussed  can  make  it  possible,  when  cells  possessing  numerous 
different  receptors  are  injected,  that  only  a  small  number  of  the  anti- 
bodies theoretically  possible  is  actually  produced.  It  is  therefore 
very  likely  that  the  immunization  of  one  animal  species  with  a  certain 
kind  of  cell  or  bacterium  results  in  the  production  of  only  part  of  the 
possible  antibodies.  When,  however,  the  same  kind  of  cell  or  bac- 
terium is  injected  into  a  second  animal  species,  it  is  highly  probable 
that  in  this  species  the  haptophore  groups  of  the  cells  will  find  a 
receptor  apparatus  which  in  part  at  least  is  different  from  that  of 
the  first  species  The  prere  quisite  for  such  a  difference  is  the  obvious 
assumption  that  the  receptor  apparatus  of  one  species  is  not  identical 
with  the  receptor  apparatus  of  a  second  not  very  closely  related 
species.  For  example,  it  is  possible  that  a  certain  haptophore  group 
a  of  the  typhoid  bacillus  finds  fitting  receptors  in  the  organism  of  the 
rabbit,  but  not  in  that  of  the  dog,  whereas  another  group,  b,  finds 
just  the  reverse  conditions.  If  these  presumptions  are  correct  an 
important  principle  for  the  practical  production  of  curative  sera  will 
follow,  namely,  that  in  any  single  case  one  would  immunize  a  number 
of  different  animal  species,  select  the  sera  containing  different  immune 
bodies,  and  by  mixing  these,  produce  a  curative  serum  containing  differ- 
ent types  of  receptors  in  as  complete  a  form  as  possible. 

Owing  to  the  importance  of  this  subject  we  have  first  under- 

1  Centralblatt  fur  Bacteriologie,  Vol.  26,  1899. 


STUDIES  ON   H.EMOLYSIXS.  93 

taken  the  experimental  study  of  the  preliminary  question  whether 
mmune  sera  derived  by  treating  two  different  animal  species  with  the 
same  cells  are  identical  so  far  as  their  antibodies  are  concerned, 
or  whether  they  are  partly  or  wholly  different.  Of  these  antibodies 
the  most  important  are  the  bacteriolytic  and  hsemolytic  immune 
bodies.  According  to  our  conception,  as  is  well  known,  these 
possess  two  haptophore  groups,  one,  the  complementophile  group  1 
and  the  other  (which  anchors  itself  to  the  receptors  of  the  cells 
causing  the  immunity)  which  we  can  briefly  designate  the  cytophile 
group.  According  to  what  has  been  said  above  it  is  this  second 
group  which  possesses  special  significance  in  the  question  under 
discussion,  and  we  may  therefore  formulate  our  problem  as  follows: 
To  determine  whether,  in  the  immunization  of  different  animal  species 
with  cells  of  one  kind,  amboceptors  (immune  bodies)  possessing  different 
cytophile  groups  arise. 

The  experimental  study  of  this  question  can  be  pursued  in  the  main 
in  two  different  ways:  1,  by  means  of  the  absorption  test  which, 
although  it  is  very  difficult,  is  applicable  to  bacteriolysins  as  well 
as  to  haBmolysins;  2,  by  neutralization  with  antiamboceptors  (anti- 
immune  bodies). 

The  latter  way,  the  more  elegant  of  the  two,  is,  however, 
presumably  applicable  only  to  those  immune  bodies  which  are 
directed  against  cells  of  the  organism.  A  hosmolytic  or  cytotoxic 
immune  body,  as  is  to  be  expected,  always  finds  points  of  attack 
in  the  organism  of  the  corresponding  animal  species,  for  this  is 
the  first  prerequisite  for  the  possibility  of  an  anti-immune  body. 
As  a  matter  of  fact  also,  such  anti-immune  bodies  have  already 
been  observed.  On  the  other  hand,  the  immune  bodies  of  bacteri- 
cidal sera,  since  their  natural  counter  groups  are  found  in  the 
bacterial  cells,  will  in  all  probability  not  find  these  groups  in  the 
cells  of  the  higher  animals.  Hence  it  seems  improbable,  unless  by 
chance  they  occur  in  an  isolated  case,  that  anti-immune  bodies 
directed  against  the  bactericidal  immune  bodies  will  be  produced. 

II.    Concerning  the  Variety  of  the  Cytophile  Groups  of  Homologous 

Immune  Bodies. 

We  selected  immunization  with  ox  blood-cells  as  being  especially 
adapted  for  these  experiments.  Such  immunization  had  already 
been  carried  out  by  von  Dungern  on  rabbits.  The  production  of 
immune  bodies  in  high  concentration  succeeds  particularly  well  in 


94 


COLLECTED  STUDIES  IN  IMMUNITY. 


this  case,  so  that  later  investigators  (Buchner,  Rehns,  Bulloch) 
have  also  employed  this  useful  combination.  In  many  cases,  most 
easily  by  means  of  intraperitoneal  injections  of  the  ox  blood,  a  potent 
hsemolysin  is  produced  of  which  0.005-0.0005  cc.  suffices  to  dissolve 
1  cc.  of  the  5%  ox-blood  mixture.  Since  the  production  of  the 
immune  body  is  unaccompanied  by  any  increase  in  complement  (as 
von  Dungern  showed  in  just  this  case)  it  is  always  necessary,  in 
order  to  bring  the  total  amount  of  immune  body  into  action,  to 
add  extra  complement.  This  is  found  in  large  amounts  in  the  serum 
of  rabbits  and  especially  in  that  of  guinea-pigs. 

Now  we  have  observed  that  the  serum  of  these  rabbits  which  had 
been  immunized  with  ox  blood  is  able  to  dissolve  not  only  the  blood- 
cells  of  oxen,  but  also  those  of  goats.  The  following  table  shows  a 
comparison  of  the  solvent  action  of  several  of  these  sera  on  the  blood- 
cells  of  oxen  and  of  goats.  Guinea-pig  serum  (0.1  or  0.15  cc.)  was 
used  as  complement  since  rabbit  serum  itself,  in  the  doses  required, 
often  exerted  a  haBmolytic  action  on  the  goat  blood-cells. 

TABLE  I. 

ACTION  OF  THE  IMMUNE  BODY  OF  THE  RABBIT  IMMUNIZED  WITH  Ox  BLOOD,  ON 
Ox  BLOOD,  AND  ON  GOAT  BLOOD. 


Ratio  of  the 

Number  of  the  Rabbit  Treated 
with  Ox  Blood. 

Complete  Solvent 
Dose  for  1  cc. 
of  Ox  Blood. 

Complete  Solvent 
Dose  for  1  cc 
of  Goat  Blood. 

Solvent  Doses 
(Approximate). 
Complete  Solvent 

Dose  for 

cc. 

cc. 

Ox  Blood  =  1. 

Jo    1  of        T-24-01  

0.0042 

0   0061 

)  -1    P; 

2 

XII-14-00  

0.0035 

0  0061 

•1  7 

3 

n_  8_oi 

0  002 

0  0035 

•1    S 

4 

H_  8-01 

0  003 

0  01 

30 

5 

I_2i-01 

0  0017 

0  0061 

3   ft 

6 

XII-17-00 

0  0014 

0  0051 

3« 

7 

XII-14-00  

0.00088 

0.0042 

:5 

8 

II-  3-01  

0.0051 

0.05 

:9.8 

9 

XII-15-00             .    . 

0  00073 

0  0073 

•10 

10 

II-  9-01  ,  

0.0035 

0.06 

:17 

This  table  shows  that  the  hsemolytic  action  of  the  immune  body 
is  always  less  on  goat  blood  than  on  ox  blood,  and  that  the  ratio 
of  the  solvent  doses  for  the  two  species  of  blood  is  not  constant  but 
varies  within  fairly  wide  limits,  as  can  be  seen  from  the  last  column. 


STUDIES  ON   H^MOLYSINS. 


95 


This  variable  ratio  indicates  that  the  solvent  action  on  the  two 
species  of  blood-cells  is  not  the  simple  function  of  one  and  the  same 
immune  body,  but  that  two  fractions  of  immune  bodies  are  pres- 
ent in  the  serum,  of  which  one  acts  exclusively  on  ox  blood-cells, 
while  the  other  fraction  acts  both  on  ox  blood  and  on  goat  blood- 
cells. 

These  relations  can  be  studied  directly  by  means  of  elective  ab- 
sorption. If  the  immune  body  is  treated  with  a  sufficient  amount  of 
ox  blood  cells  and  the  fluid  is  then  separated  by  centrifuge,  it  will 
be  found  that  the  serum  has  lost  its  solvent  action  for  both  species 


FIG.  l. 


Ox 


Blood-cell  of  an  ox  and  of  a  goat,  showing  specific  and  common  receptors 


of  blood;  for  by  means  of  the  ox  blood-cells,  which  as  the  original 
excitants  of  the  immunity  are  carriers  of  all  the  receptors  in  question, 
both  fractions  of  immune  body  have  been  bound.  When  the  same 
experiment  is  performed  with  goat  blood-cells,  it  can  be  shown  that 
the  fluid  /«zs  lost  its  solvent  power  for  goat  blood,  while  that  for  ox  blood 
remains.  In  favorable  cases  the  solvent  power  for  ox  blood  may 
remain  almost  unchanged.  The  conditions  present  can  be  readily 
understood  by  reference  to  Fig.  1. 

Let  this  represent  schematically  three  portions  of  the  combin- 
ing groups  of  the  blood-cells,  of  which  the  first,  a,  is  present  only  in 


COLLECTED   STUDIES  IN  IMMUNITY. 


the  ox  blood-cells,  the  second,  7-,  only  in  goat  blood-cells,  and  the 
third,  /?,  in  both.  If  a  rabbit  is  injected  with  ox  blood,  the  ambo- 
ceptors  (immune  bodies)  corresponding  to  groups  a  and  /?  will  be 
formed.  Ox  blood-cells,  by  means  of  their  a.  and  /?  groups,  will 
then  be  able  to  anchor  all  the  immune  bodies,  whereas  goat  blood- 
cells  will  anchor  only  the  immune  body  of  portion  /?,  leaving  the 
immune  body  of  portion  a.  in  the  solution. 

If,  as  this  explanation  assumes,  the  goat  blood-cells  possess  a 
certain  portion  ofr  eceptors  which  are  common  to  goat  and  ox  blood- 
cells,  it  is  essential  that  by  treating  rabbits  with  goat  blood  an  immune 
body  should  be  obtained  which  likewise  would  act  on  both  species 
of  blood.  This,  in  fact,  is  the  case.  And  here,  as  in  the  first  case, 
the  solvent  power  for  the  two  species  of  blood-cells  usually  differs, 
though  of  course  the  relations  are  reversed  from  those  in  that  case, 
.as  can  be  seen  by  reference  to  Table  II. 

TABLE  II. 

ACTION  OF  THE  IMMUNE  BODY  OF  THE  RABBIT  WHICH  HAD  BEEN  TREATED  WITH 
GOAT  BLOOD,  ON  GOAT  BLOOD,  AND  Ox  BLOOD. 

(Reactivation  with  guinea-pig  serum. ) 


Ratio  of  the 

Number  of  the  Rabbit  Treated 

Complete  Solvent 
Dose  for  1  cc. 

Complete  Solvent 
Dose  for  1  cc.  of 

Solvent  Doses 
(Approximate). 

with  Goat  Blood. 

of  Goat  Blood. 

Ox  Blood. 

Complete  Solvent 

Dose  for  Goat 

cc. 

cc. 

Blood  =  1. 

No  1  of     11-28-01         

0   01 

0  024 

1-2  4 

"    2  "        1-14-01       

0   0061 

0  025 

1:4 

"    3  "      II-  7-01  l  

0.0012 

0  025 

1:20 

"    4  "  XII-18-00       

0.0071 

0  25 

1:<33 

(almost  complete) 

1  On  employing  the  same  serum  on  a  different  ox  blood,  0.05  cc.  produced 
no  solution  at  all,  and  0.1  cc.  merely  a  trace.  This  is  evidently  due  to  a  casual, 
individual  lack  of  receptors  in  the  ox  blood-cells  in  question,  such  as  showed 
itself  so  frequently  in  goat  blood  when  we  studied  isolysins. 

Because  of  these  ratios  we  shall  have  to  assume  that  the  goat 
blood-cells  in  this  case  possess  a  second  system  of  binding  groups 
which  is  peculiar  to  them  and  represented  in  the  above  diagram 
by  f.  They  possess  these,  of  course,  in  addition  to  the  receptors,  p 
which  they  have  in  common  with  the  ox  blood-cells.  In  accordance 
with  this,  in  the  elective  absorption  test  in  this  case,  the  goat  blood- 
cells  will  bind  the  entire  lot  of  immune  bodies;  whereas  when  ox 


STUDIES   OX   H.EMOLYSINS.  97 

blood-cells  are  used,  the  group  7-  will  be  left  behind,  for  this  possesses 
affinity  only  for  the  goat  blood-cells. 

The  following  protocol  shows  the  results  of  two  series  of  experi- 
ments, which  exhibits  the  effect  of  such  reciprocal  binding: 

To  each  5  cc.  of  a  o^J  mixture  of  ox  blood-cells  or  goat  blood- 
cells  (freed  from  serum  by  centrifuge)  varying  amounts  of  the  immune 
body  of  a  rabbit  which  had  been  immunized  with  ox  blood  are 
added.  The  amount  of  immune  body  is  seen  in  the  first  column 
of  the  table;  in  the  second  and  third  columns  the  complete  solvent 
doses  (for  ox  blood  and  for  goat  blood)  contained  in  each  specimen 
are  given,  as  they  were  determined  by  tests  made  at  the  same  time. 
The  mixtures  are  made  up  to  6  cc.  with  physiological  salt  solution, 
kept  at  37°  C.  for  1J  hours  and  then  centrifuged.  Two  equal  por- 
tions of  each  of  the  decanted  fluids  are  then  taken  and  again  mixed 
with  corresponding  amounts  of  blood-cells.  Finally  guinea-pig 
serum  is  added  to  activate  the  mixtures.  The  haemolytic  action 
which  the  decanted  portions  exerted  on  ox  blood-cells  and  on  goat 
blood-cells  is  seen  in  the  table.  (See  Table  III.) 

The  union  of  the  immune  body  with  the  ox  blood-cells  has  resulted 
in  a  considerable  abstraction  of  both  portions  of  immune  body.  On 
the  other  hand,  the  union  with  goat  blood-cells,  by  which  the  action 
of  the  fluid  is  considerably  decreased  for  goat  blood-cells,  causes  very 
little  decrease  in  the  solvent  power  for  ox  blood. 

In  contrast  to  this  experiment  we  here  reproduce  an  analogous 
experiment  which  shows  a  directly  opposite  behavior  of  the  two 
fractions  of  immune  body  of  a  rabbit  immunized  with  goat  blood. 
(See  Table  IV.) 

Here  the  goat  blood-cells  bind  both  portions  of  the  immune  body, 
while  after  treatment  with  ox  blood-cells  the  fraction  acting  on  goat 
blood  is  left  almost  intact. 

Hence  by  means  of  this  crossed  immunization  and  reciprocal  elec- 
tive absorption  we  succeed  in  demonstrating  that  in  the  case  of  the 
rabbits  treated  respectively  with  goat  blood  and  ox  blood  two 
large  fractions  of  immune  bodies  can  be  separated.  Of  these,  one 
fraction  is  common  to  both  sera;  the  other  is  peculiar  to  each  of 
them.  The  main  groups  of  receptors  shown  in  the  above  illustra- 
tion and  designated  a  and  ft  for  ox  blood,  and  ft  and  7-  for  goat  blood, 
are  thus  to  be  differentiated. 

We  have  deemed  it  important  to  supplement  this  analysis  by 
experiments  on  a  second  species  of  animal,  and  have  therefore  treated 


COLLECTED  STUDIES  IN  IMMUNITY. 


a  goat  with  ox  blood.  Naturally  the  serum  of  the  goat  so  treated 
dissolves  ox  blood-cells.  Besides  this,  however,  it  manifests  the 
ability  to  dissolve  the  blood-cells  of  a  few  other  goats,  and  therefore 
contains  true  isolysins  such  as  we  have  previously  produced  by 
treating  goats  with  goat  blood.  Thus  0.025  cc.  of  the  serum  of 

TABLE  III. 

BINDING  OF  THE  IMMUNE  BODY  OF  A  RABBIT  TREATED  WITH  Ox  BLOOD,  WITH 
Ox  BLOOD-CELLS,  AND  GOAT  BLOOD-CELLS. 


Solvent  Power  of  the  Decanted  Fluids. 

Amount  of  the 

Number  of  Solv- 
ent Doses  Con- 

Body Added. 
(Derived  from 

tained  Therein. 

A  ,  after  Binding  with 
Ox  Blood. 

B,  after  Binding  with 
Goat  Blood. 

a  Rabbit  by 

Treating  with 

Ox  Blood.) 

(a)  For 
Ox 
Blood. 

(6)  For 
Goat 
Blood. 

(a)  On 
Ox  Blood. 

(6)  On 
Goat  Blood. 

(a)  On 
Ox  Blood. 

(6)  On 
Goat  Blood. 

No.         cc. 

1       0.001 

* 

* 

0 

0 

0 

0 

2      0.002 

* 

A 

0 

0 

trace 

0 

3      0.003 

| 

A 

0 

0 

very  little 

0 

4      0.004 

f 

2 

0 

0 

very  little 

0 

to  little 

5      0.005 

1 

1 

0 

0 

mod.  to  little 

0 

6     0.006 

1 

ft 

0 

0 

moderate 

0 

7     0.007 

H 

* 

0 

0 

i  i 

0 

8     0.008 

ll 

0 

0 

alm'st  comp. 

0 

9     0.01 

if 

i 

0 

0 

complete 

0 

10     0.012 

2 

i 

0 

0 

ft 

faint  trace 

11      0.016 

2§ 

•i 

0 

0 

14 

faint  trace 

12     0.02 

3* 

faint  trace 

very  little 

(t 

very  little,top 

13     0.024 

4 

i* 

very  little 

1  1       1  1 

tl 

little,  top 

14     0.032 

61 

if 

lit.  to  mod. 

little  to 

1  1 

little 

very  little 

15     0.048 

8 

2| 

f  C 

little 

1  1 

i  i 

16     0.06 

10 

3 

alm'st  comp. 

moderate 

(  { 

t  ( 

17     0.08 

13| 

4 

complete 

fair 

1  1 

i  ( 

18     0.1 

16§ 

5 

complete 

strong  to  al- 

( ( 

little  to  mod. 

i  ( 

most  comp. 

39     0.14 

23£ 

7 

(  ( 

complete 

(  t 

mod.  to  little 

one  of  our  goats,  on  the  addition  of  complement,  dissolved  the  usual 
amount  of  ox  blood-cells.  This  serum,  however,  dissolved  but  two 
out  of  five  different  specimens  of  goat  blood,  and  the  isolysin  con- 
stituent was  present  in  only  very  small  amounts,  so  that  it  required 
0.75  cc.  serum  (thirty  times  the  above  amount)  to  effect  complete 
haemolysis  of  sensitive  goat  blood-cells.  Hence  in  this  case  also 
the  development  of  all  such  amboceptors  as  could  find  a  point  of 


STUDIES  ON  H^EMOLYSINS. 


attachment  (receptor)  in  the  blood-cells  of  the  individual  goat  itself 
has  been  avoided,  and  the  phenomenon  which  we  have  previously 
designated  as  a  "horror  autotoxicus  " l  is  again  presented. 

TABLE  IV. 

BINDING  OF  THE  IMMUNE  BODY  OP  A  RABBIT  IMMUNIZED  WITH  GOAT  BLOOD, 
ON  Ox  BLOOD  AND  GOAT  BLOOD-CELLS. 


Number  of 

Solvent  Power  of  the  Decanted  Fluids. 

Amount  of 
the  Immune 
Body  Added. 
(Derived  from 

Solvent  Doses 
Contained 
Therein. 

A.  After  Binding  with 
Ox  Blood. 

B.  After  Binding  with 
Goat-Blood. 

a  Rabbit  by 

Treating  with 
Goat  Blood.) 

(a)  For 
Ox 
Blood. 

(6)  For 
Goat 
Blood. 

(a)  For 
Ox  Blood. 

(6)  For 
Goat  Blood 

(a)  For 
Ox  Blood. 

(6)  For 
Goat  Blood. 

No.        cc. 

1      0.038 

T45 

1 

0 

fair  to  mod- 

0 

0 

erate 

2     0.05 

if 

i 

0 

almost  complete 

0 

0 

3     0.062 

1ft 

0 

complete 

0 

0 

4     0.074 

i 

2 

0 

0 

0 

5     0.1 

« 

2| 

0 

0 

minimal  trace 

6     0.13 

3i 

0 

0 

trace 

7     0.15 

4 

0 

0 

1  t 

8     0.2 

5 

0 

0 

very  little 

9     0.25 

2 

<* 

0 

0 

little 

10     0.3 

8 

0 

0 

n 

11      0.38 

3 

10 

0 

0 

fair  to  strong 

From  this  experiment  we  can  at  once  conclude  that  this  receptor 
system  /?  actually  consists  of  different  components,  of  which  only 
those  separate  amboceptors  (immune  bodies)  are  found  in  the  serum 
of  goats  treated  with  ox  blood  whose  receptors  are  absent  in  the 
blood-cells  of  the  goat  itself. 

The  most  important  result  of  these  investigations — investigations 
complete  in  themselves — is  this :  By  treating  animals  with  ox  blood,  two 
fractions  of  immune  bodies  are  formed,  of  which  one  acts  only  on  ox  blood 
and  the  other  also  on  goat  blood;  whereas  by  treatment  with  goat  blood 
the  contrary  though  entirely  analogous  result  ensues.  These  two  frac- 

1  We  were  also  able  to  observe  that  the  immune  body  of  the  rabbits  which 
had  been  immunized  with  ox  blood  and  goat  blood  acted  also  on  sheep  blood. 
Closer  investigation  would  probably  show  that  this  behavior  is  analogous  to 
its  action  on  goat  blood.  This  corresponds  entirely  to  our  earlier  observations 
on  the  extensive  similarity  of  the  receptor  apparatus  of  goat  and  sheep  blood 
us  it  was  manifested  particularly  by  the  experiments  on  isolysins. 


100  COLLECTED  STUDIES  IN   IMMUNITY. 

tions  do  not  correspond  to  two  different  single  immune  bodies,  but  each 
fraction  includes  several,  perhaps  an  entire  host  of  immune  bodies. 

The  experiments  also  lead  to  conclusions  of  considerable  impor- 
tance in  another  direction,  namely,  as  affecting  our  conception  of 
cellular  specificity  and  of  the  specificity  of  reaction  products.  '  Here- 
tofore it  has  been  held  that  the  injection  of  blood  of  species  a  results 
in  a  specific  immune  serum,  i.e.  one  acting  only  on  a;  and  even 
Metchnikoff 1  has  recently  expressed  this  view.  We  had  already 
become  acquainted  with  certain  exceptions  to  this  principle.  The 
isolysin,  for  example,  produced  by  injecting  goats  with  goat  blood, 
also  dissolves  sheep  blood;  and,  vice  versa,  the  immune  body  of 
goats  which  have  been  injected  with  sheep  blood  acts  also  as  an 
isolysin.  At  that  time  we  emphasized  that  these  results  are  only 
to  be  explained  by  assuming  that  certain  types  of  receptors  are  com- 
mon to  both  species  of  blood.  The  same  holds  true  in  the  case 
under  discussion,  von  Dungern2  has  come  to  the  same  conclusion 
as  a  result  of  his  experiments.  He  found  that  the  immune  body 
produced  by  injection  of  ciliated  epithelium  acts  also  on  the  blood- 
cells  of  the  same  species,  and  that  conversely  the  hsemolytic  immune 
body  produced  by  injection  of  blood-cells  is  partially  bound  by 
ciliated  epithelium. 

All  these  circumstances  indicate  that  we  must  not  regard  the  spe- 
cificity of  the  immune  bodies  from  the  conception  of  specificity  employed 
in  systematic  zoology  and  botany.  The  immune  sera,  as  we  have  often 
mentioned,  are  not  of  simple  Unitarian  nature,  but  consist  of  a  series 
of  single  immune  bodies  whose  cytophile  haptophore  groups  cor- 
respond to  the  receptors  of  the  exciting  cells.  Hence  such  an  immune 
serum  will  be  able  to  affect  all  such  elements  which  possess  any  one  of 
the  receptors  whose  type  is  common  to  those  elements  and  the  original 
cell  "a."  The  influence  exerted  by  the  immune  serum  will  be  power- 
ful in  proportion  to  the  extent  of  this  correspondence  of  receptors. 
Now  we  have  reason  to  believe  (cf.  Ehrlich's  deductions,  1.  c.,  and 
Weigert's  in  Lubarsch-Oster  tag's  Ergebnisse  der  Pathologic,  1887,  p. 
141)  that  certain  receptors  are  very  widely  distributed  among  various 
animal  species.  Thus  the  blood-cells  of  a  large  number  of  species 
possess  receptors  fitting  ricin,  abrin,  crotin,  and  tetanolysin,  and  gan- 
glion cells  of  the  most  divergent  animals  possess  receptors  for  tetano- 

1  Revue  generate  des  sciences,  1901,  No.  1. 

2  See  page  47. 


STUDIES  ON   ELEMOLYSINS.  101 

spasmin  or  for  sausage  poison.  Within  the  animal  organism,  in  like 
manner,  certain  receptors  are  evidently  widely  distributed  in  the 
most  varied  organs,  as  is  shown,  for  example,  by  the  experiments 
with  tetanus  poison.  Looked  at  from  this  standpoint,  the  apparent 
deviations  in  specificity  are  comprehensible.  We  are  convinced 
that  in  this  field  the  near  future  will  furnish  us  with  extensive  ma- 
terial of  immense  value  in  the  analysis  and  study  of  the  distribution 
of  receptors.  We  are  led  to  conclude,  therefore,  that  in  the  produc- 
tion of  immune  bodies  by  immunizing  with  cells  we  can  speak  of 
specificity  only  in  the  sense  that  there  is  always  a  specific  relation 
between  the  separate  types  of  immune  bodies  and  the  receptors. 


The  foregoing  experiments  constitute  conclusive  proof  of  the 
plurality  of  the  immune  bodies  produced  by  injections  of  ox 
blood  and  goat  blood.  We  next  endeavored  to  extend  these  results 
by  effecting  a  differentiation  of  various  groups  of  immune  bodies 
by  means  of  the  anti-immune  bodies.1  The  highest  concentration  of 
immune  bodies  at  our  disposal  was  the  serum  of  a  rabbit  which  had 
been  immunized  with  ox  blood.  For  various  reasons  we  chose  goats 
for  these  immunizing  experiments,  for  we  knew  that  their  blood- 
cells  already  contained  receptors  capable  of  binding  a  portion  of  the 
mixed  immune  bodies.  In  treating  these  goats  we  used  the  inactive 
serum  of  a  rabbit  immunized  with  ox  blood.  This  serum,  which 
was  of  the  highest  possible  strength,  was  injected  subcutaneously. 
During  the  course  of  two  months  we  had  thus  injected  120  cc.  of 
an  immune  body  serum,  of  which  0.005  cc.  sufficed,  when  reactivated 
with  guinea-pig  serum,  to  completely  dissolve  1  cc.  of  a  5%  mixture 
of  ox  blood-cells.  At  the  end  of  that  time  we  were  able  to  demon- 
strate the  existence  of  an  anti-immune  body  of  considerable  pro- 
tective power.  That  this  was  really  an  anti-immune  body  which 
inhibited  the  anchoring  of  the  immune  body  to  the  red  blood-cells, 
is  seen  by  the  following  combining  experiment. 

0.5  cc.  of  the  anti- immune  body  (inactive  serum  of  a  goat  treated 
as  just  described)  are  mixed  with  varying  amounts  of  the  immune 
body  (inactive  serum)  of  a  rabbit  treated  with  ox  blood.  Thereupon 
1  cc.  of  a  5%  mixture  of  blood-cells  is  added  to  each  specimen.  These 
are  then  kept  at  40C  C.  for  one  hour  and  centrifuged.  The  various 
sediments  are  then  mixed  with  salt  solution  and  0.15  cc.  normal 
guinea-pig  serum.  A  parallel  experiment  (control  test)  is  made  in 

1  See  Ehrlich's  recent  study,  page  573. 


102 


COLLECTED  STUDIES  IN  IMMUNITY. 


which  the  anti-immune  body  is  replaced  by  the  same  amount  (0.5  cc.) 
of  inactive  normal  goat  serum.  The  degree  of  solution  is  shown  in 
Table  V. 

TABLE  V. 


Number  of 

Amount  of  Immune 
Body  Added. 

Complete 
Solvent 
Doses 

Degree  of  Solution  After 
Addition  of  Complement. 

Degree  of  Solution 
of  the  Sediment  in 
the  Control  Test. 

cc. 

Therein. 

lo.    1     0.00125 

1 

no  solution 

complete  solution 

2     0.0025 

2 

n        « 

3     0.00375 

3 

e  (            (( 

4     0.005 

4 

trace  solution 

5    0.0075 

6 

little  solution 

6    0.01 

8 

almost  complete  solution 

7    0.025 

20 

complete  tolution 

From  these  figures  we  see  that  a  single  solvent  dose  becomes 
available  for  combination  with  the  red  blood-cells  only  after  eight 
times  the  solvent  dose  has  been  added,  and  that  a  triple  dose  is  com- 
pletely neutralized,  i.e.,  prevented  from  combining  with  the  blood- 
cell.  The  control  test  shows  that  0.5  cc.  of  a  normal  inactive  goat 
serum  does  not  prevent  the  combination  of  a  single  solvent  dose  of 
mmune  body  (0.00125  cc.).  The  sediment  in  this  case  is  competely 
dissolved  on  the  addition  of  complement.1  By  this  experiment  the 
inhibiting  substance  is  definitely  characterized  as  an  anti-immune 
body.  The  following  example  will  show  the  exact  quantitative 
relation  of  this  anti-immune  body. 

Each  0.4  cc.  inactivated  serum  (anti-immune  body)  of  the  goat 
treated  with  immune  body  are  mixed  with  the  given  amount  of 
inactive  serum  (immune  body)  of  a  rabbit  treated  with  ox  blood. 
The  specimens  are  made  up  to  the  same  volume  by  the  addition  of 
salt  solution,  kept  at  room  temperature  for  half  an  hour,  and  then 
mixed  with  1  cc.  of  a  5%  suspension  of  ox  blood,  and  with  0.15  cc. 
normal  guinea-pig  serum  (complement).  A  control  test  is  made  in 
which  normal  inactive  goat  serum  is  used  instead  of  the  anti-immune 
body.  (See  Table  VI.) 

1  We  should  like  to  remark  that  in  the  course  of  numerous  experiments 
Tve  have  now  and  then  found  normal  goat  sera  containing  slight  amounts 
of  an  anti-immune  body  acting  on  the  immune  body  of  rabbits  treated  with 
ox  blood.  This  is  to  be  brought  into  connection  with  the  law  (see  also  Neisser- 
1.  c.)  that  the  artificially  produced  antibodies  frequently  represent  only  an 
increase  of  normal  functions. 


STUDIES  OX   H-EMOLYSINS. 
TABLE  VI. 


103 


Experiment  with  0.4  cc.  Anti-immune  Body. 

Control  Test  with  0.4  cc.  Normal 
Goat  Serum. 

Amount  of 

Amount  of 

Immune 
Body. 

Solvent  Action. 

Immune 
Body.    . 

Solvent  Action. 

cc. 

cc. 

0.0175 
0.0145 

complete  solution 
strong 

0.001 
0.00085 

complete  solution 
almost  complete  solution 

0.012 

fairly  strong  solution 

0.0007 

strong  solution 

0.01 

moderate  solution 

0.0006 

it            1  1 

0.0085 

little  solution 

0.0005 

moderate  solution 

0.007 

1  1          i  ( 

0.006 

trace  solution 

0.005 

small  trace  solution 

0.0044 

(  (            (  e               it 

0.00375 

it            (i               (  f 

0.003 

minimal  trace  solution 

0.0025 

(i           K           tt 

0.002 

t<           ii           (( 

0.0018 

0 

This  shows  that  0.2  cc.  of  the  anti-immune  body  are  able  to  com- 
pletely inhibit  the  action  of  1.8  times  the  solvent  dose  of  immune 
body  as  determined  by  the  control  test,  and  that  it  almost  neutral- 
izes the  action  of  five  times  such  a  dose.  If,  however,  we  measure 
the  protective  power  by  comparing  the  complete  solvent  doses  in 
the  two  cases,  this  appears  much  stronger.  The  ratio  of  the  com- 
plete solvent  doses  in  the  presence  of  immune  body  and  in  the  control 
test  is  then  1:17.5.  We  shall  discuss  the  reason  for  this  later. 

Since  the  inactive  rabbit  serum  employed  in  immunization  con- 
tained complementoids,  the  presence  of  anticomplements  along  with 
the  anti-immune  body  is  easily  understood.  The  anticomplements  at 
first  were  directed  against  rabbit  serum.  After  the  immunization 
had  continued  for  some  time  longer  anticomplements  appeared 
directed  against  certain  complements  of  guinea-pig  serum.  In  these 
experiments,  therefore,  in  order  to  overcome  the  anticomplement 
action  (in  reality  insignificant)  directed  against  the  reactivating 
guinea-pig  serum,  it  was  merely  necessary  to  employ  a  considerable 
excess  of  the  latter. 

In  contrast  to  these  results  are  those  obtained  in  an  analogous 
series  of  experiments,  in  which,  however,  the  complement  was  in  the 
form  of  goat  serum  instead  of  guinea-pig  serum.  (See  Table  Via.) 


104 


COLLECTED   STUDIES  IN  IMMUNITY. 


TABLE  Via. 


Experiment  with  0.4  cc.  Anti-immune  Body. 

Control  Test  with  0.4  cc.  Normal 
Goat  Serum. 

Amount  of 

Amount  of 

Immune 

Solvent  Action. 

Immune 

Solvent  Action. 

Body. 

Body. 

cc. 

cc. 

- 

0.051 
0.042 
0.029 

complete  solution 
almost  complete  solution 
moderate  solution 

0.051 
0.042 
0.029 

complete  solution 
almost  complete  solution 
moderate  solution 

0.02 

trace  solution 

0.02 

very  little  solution 

0.017 

faint  trace  solution 

0.017 

trace  solution 

0.014 

0 

0.014 

0 

In  this  combination  the  anti-immune  body  exerts  no  action.  Hence 
we  must  here  be  dealing  with  a  particular  type  of  immune  body  which 
effects  a  combination  with  a  complement  present  in  goat  serum.  This 
immune  body  enters  into  no  relation  with  the  complex  of  immune 
bodies  here  present;  it  must  therefore  possess  a  haptophore  group 
which  finds  no  fitting  counter  group  therein. 

As  a  matter  of  fact  the  completion  by  means  of  goat  serum  occu- 
pies a  special  position,  for  the  quantitative  relations  of  the  immune 
body  are  entirely  different  from  those  observed  when  guinea-pig 
serum  is  used.  In  order  to  effect  complete  solution  when  goat  serum 
is  used  as  complement,  it  is  necessary,  as  a  rule,  to  use  from  ten  to 
thirty  times  the  amount  of  immune  body  that  would  be  required 
if  guinea-pig  serum  were  used  as  complement.  This  is  well  shown 
by  Table  VII. 

TABLE  VII. 


Complete  Solvent  Dose 

Compete  Solvent  Dose 

No. 

of  Immune  Body  when 
Complemented  with 
Guinea-pig  Serum  0.15. 

of  Immune  Body  when 
Complemented  with 
Goat  Serum  0.5. 

Ratio  of  the  Two  Doses. 

cc. 

cc. 

1 

0.005 

0.05 

1:10 

2 

0.0075 

0.075 

1:10 

3 

0.0075 

0.1 

1:13 

4 

0.0025 

0.075 

1:30 

That  this  behavior  is  not  due  to  a  smaller  content  of  complement 
in  the  goat  serum  can  readily  be  determined  by  suitable  experiments 
especially  by  increasing  the  dose  of  the  latter. 


STUDIES  ON  HJEMOLYSINS.  105 

This  can  only  be  explained  by  assuming  that  only  part  of  the  total 
number  of  immune  bodies  find  fitting  complements  hi  goat  serum, 
and  that  this  partial  number  varies,  but  is  always  less  than  the  number 
of  immune  bodies  activated  by  guinea-pig  serum.  The  diagram 
presented  below  will  best  make  this  relation  clear. 

We  have  made  a  further  series  of  experiments  in  order  to  com- 
plete these  studies,  and  have  discovered  that  our  anti-immune  body 
also  protected  goat  blood-cells  against  the  immune  body  derived 
from  a  rabbit  treated  with  ox  blood.  This  of  course  is  quite  natural, 
for  we  have  already  shown  that  this  action  on  a  strange  species  of 
blood  depends  on  a  concordance  of  certain  haptophore  groups. 
Similarly,  the  anti-immune  body  protects  ox  blood-cells  against  the 
immune  body  of  a  rabbit  immunized  with  goat  blood. 

These  experiments  lead  us  to  the  following  conclusions:  The 
anti-immune  body  derived  by  injecting  goats  with  immune  bodies  of 
rabbits  is  not  a  simple  uniform  [einheitlich]  substance,  but  is  made  up 
of  a  whole  series  of  partial  immune  bodies.  In  the  ox  blood  used  to 
immunize  the  rabbits  we  have  already  distinguished  two  main  portions 
of  receptors  to  which  again  two  main  portions  of  the  resulting  immune 
body  correspond.  Each  of  the  latter  portions  in  all  probability  con- 
tains a  host  of  partial  immune  bodies,  and  we  must  assume  thatf 
corresponding  to  this,  the  anti-immune  bodies  also  possess  a  complex 
constitution. 

In  the  following  diagram  it  is  not  sought  to  express  that  the 
immune  bodies  which  can  be  activated  by  guinea-pig  serum  are  all 
identical.  On  the  contrary  each  group  may  represent  a  different 
kind  of  immune  body. 

We  have  seen  that  there  is  a  great  difference  between  the  dose 
of  immune  body  which  is  completely  neutralized  by  a  certain  amount 
of  anti-immune  body,  and  that  which  in  the  presence  of  anti-immune 
body  causes  complete  solution.  This  can  be  understood  when  we 
recall  the  above-mentioned  distribution  of  partial  immune  bodies, 
and  examine  the  diagram,  Fig.  2. 

In  order  to  choose  a  simple  illustration  let  us  assume  that,  corre- 
sponding to  the  diagram,  the  immune  serum  of  the  rabbit  immu- 
nized with  ox  blood  contains  only  two  different  types  of  immune 
bodies  and  these,  furthermore,  in  unequal  amounts.  Let  the  main 
portion  be  represented  by  immune  body  type  a,  which  is  activated 
by  a  particular  complement  present  in  the  animaPs  own  (rabbit's) 
serum.  Further,  let  the  second  portion,  present  in  much  less  amount, 


106  COLLECTED  STUDIES  IX   IMMUNITY. 

be  represented  by  immune  body  type  b,  which  is  activated  by  a 
different  complement  also  present  in  rabbit  serum  but  found  in 
goat  serum  as  well.  Let  the  proportion  of  a:  b  be  as  10: 1 ;  i.e.,  a  quan- 
tity of  immune  serum  containing  one  complete  solvent  dose  of  b 
will  contain  ten  solvent  doses  of  a.  In  this  case  then  it  will  require 
ten  times  as  much  of  the  immune  serum  to  effect  complete  solution 
by  means  of  immune  body  6  (which  is  the  case  when  goat  serum, 
which  contains  complement  only  for  b,  serves  for  reactivation)  used 
when  immune  body  a  is  employed.  The  composition  of  this  immune 
,serum  can  be  represented  by  the  formula  10a+16. 

/ 

FIG.  2. 


^H  "tibkf  4kg^  ttjW  ^^/  \*UL/  ^M&  t&to  iiifc'  ^M       ^fc4/k 


Diagram  to  show  the  two  types  of  immune  bodies  present  in  the  immune 
serum  of  a  rabbit  treated  with  ox  blood.  Each  immune  body  symbol  corre- 
sponds to  one  solvent  dose  for  the  amount  of  ox  blood  employed  in  the  ex- 
periment. Immune  body  type  a  is  present  in  ten  times  the  amount  of  type  6. 
The  complementophile  groups  of  a  and  b  differ;  hence  also  the  complements 
differ.  The  anti-immune  body  serum  possesses  anti-immune  bodies  only 
against  a. 

As  is  seen  by  the  experiments,  an  anti-immune  body  exists  only 
against  immune  body  type  a.  If  therefore  to  an  amount  of  immune 
body  which  contains  one  solvent  dose  of  immune  body  b  and  ten 
solvent  doses  of  immune  body  a  (i.e.,  10a+ 16)  a  large  quantity  of  anti- 
immune  body  serum  is  added,  and  then  sufficient  suitable  complement 
it  will  be  found  that  solution  always  occurs,  for  the  reason  that  a 
single  solvent  dose  of  b  is  present  which  is  unaffected  by  the  anti- 
immune  body  although  this  was  able  to  neutralize  ten  solvent  doses 
of  a.  One-tenth  of  the  above  amount  of  immune  body,  on  the  other 


STUDIES  ON  BLEMOLYSINS.  107 

hand,  will  be  completely  inhibited  in  its  action.  For  this  contains 
one  complete  solvent  dose  of  immune  body  a  which  is  neutralized 
by  the  anti-immune  body,  and  only  one-tenth  of  a  solvent  dose  of  b 
which,  although  not  affected  by  the  anti-immune  body,  is  so  slight 
as  not  to  cause  any  appreciable  solution.  Only  when  larger  amounts 
of  immune  body  are  employed  in  which  b  becomes  active  does  solution 
occur,  and  this  becomes  complete  only  when  that  quantity  is  reached 
which  contains  10a+16.  Naturally,  if  the  ratio  is  1:20,  a  quantity 
will  be  required  which  can  be  represented  by  the  formula  20a+16. 

These  explanations  will  perhaps  suffice  to  make  the  above-men- 
tioned peculiarities  in  the  action  of  the  anti-immune  body  com- 
prehensible. They  will  perhaps  also  make  clear  that  between  the 
dose  of  immune  body  whose  action  is  completely  inhibited  by  the  anti- 
immune-body  serum  and  the  dose  which  causes  complete  solution  a  large 
number  of  intermediary  stages  exist  in  which  the  degree  of  solution 
gradually  increases. 

In  reality  the  circumstances  are  much  more  complicated  than 
this ;  for  with  the  increase  in  the  dose  of  immune  body  a  large  number 
of  new  immune  bodies,  similarly  superposed,  come  into  action — 
immune  bodies  which  find  few  or  no  corresponding  anti-immune 
bodies  in  the  antiserum. 

This  brings  us  to  another  important  question:  7s  it  possible  by 
means  of  the  anti-immune  body  to  demonstrate  the  difference  of  the 
immune  bodies  produced  by  injections  of  ox  blood  into  different  species  ? 

Our  first  experiments  were  undertaken  with  the  serum  of  goats 
which  had  been  immunized  with  ox  blood.  As  will  be  seen  by  the 
following  figures,  our  anti-immune  body  (derived  by  injections  of 
an  immune  body  obtained  from  rabbits)  hi  this  case  exerts  no  action. 
The  varying  amounts  of  immune  body  mentioned  are  mixed  with 
0.4  cc.  anti-immune  body  and  then  with  1  cc.  5%  ox  blood  suspension 
and  0.5  cc.  normal  active  goat  serum  to  activate  the  mixture.  In 
the  control  test  0.4  cc.  inactive  normal  goat  serum  are  used  instead 
of  the  anti-immune  body.  (See  Table  VIII.) 

That  this  serum  differed  markedly  with  respect  to  its  content  of 
individual  immune  bodies  was  already  shown  by  the  fact  that,  in 
contrast  to  the  serum  of  immunized  rabbits,  it  did  not  possess  a 
hsemolysin  acting  on  all  goat  blood-cells  in  general,  since  such  a  hsemoly- 
sin  would  have  exerted  a  most  injurious  action  in  the  form  of  an  auto- 
lysin.  As  a  matter  of  fact,  the  law  already  mentioned  under  the  name 
*l  horror  autotoxicus  "  applied  here  also,  and  hence  merely  an  isolysin 


108 


COLLECTED  STUDIES  IN  IMMUNITY. 


was  developed  which  acted  only  on  goat  blood-cells  of  a  few  individuals 
and  which  therefore  possessed  only  a  few  individual  special  groups 
in  its  immune  bodies.  Against  this  isolysin,  which  represented  a 
relatively  small  portion  of  the  types  of  immune  bodies  found  in  the 
goat,  our  anti-immune  body  also  proved  entirely  ineffective,  as  is 
seen  in  Table  IX. 

TABLE  VIII. 


Experiment  with  Anti-immune  Body. 

Control  Test. 

Amount 
of  Immune 
Body. 
cc. 

Solvent  Action. 

Amount 
of  Immune 
Body, 
cc. 

Solvent  Action. 

0.051 
0.042 
0.035 
0.029 
0.02 
0.017 
0.014 

complete  solution 
almost  complete  solution 
strong  solution 
moderate  solution 
trace  solution 
doubtful 
0 

0.051 
0.042 
0.035 
0.029 
0.02 
0.017 
0.014 

complete  solution 
almost  completely  dissolved 
almost  dissolved 
moderate  solution 
very  little  solution 
trace  solution 
0 

In  this  experiment  the  method  was  exactly  similar  to  that  of  the 
previous  ones.  The  blood  was  from  goat  No.  Ill,  1  cc.  of  a  5% 
suspension  being  used. 

TABLE  IX. 


Experiment  with  0.4  cc.  Anti-immune 
Body. 


Control  Test  with  0.4  cc.  Normal  Inactive 
Goat  Serum. 


Amount  of 

Amount  of 

Immune  Body. 

Solvent  Action. 

Immune  Body. 

Solvent  Action. 

cc. 

cc. 

1.5 

complete 

1.5 

complete 

1.0 

strong 

1.0 

strong 

0.88 
0.61 

strong 
moderate 

0.88 
0.61 

strong 
moderate 

0.51 

little 

0.51 

little 

0.42 

trace 

0.42 

trace 

0.35 

0 

0.35 

0 

We  see  from  this  that  by  treating  a  goat  with  ox  blood-cells,  immune 
bodies  have  been  formed  the  main  portion  of  which  differs  from  those 
obtained  by  immunizing  rabbits  with  ox  blood  or  goat  blood. 

A  second  species  of  animal  in  which  we  have  been  able  to  demon- 
strate a  difference  in  the  immune  bodies  is  the  goose.  The  immune 
bodies  obtained  by  injecting  a  goose  with  ox  blood-cells  are  also  not  in 


STUDIES   ON  ILEMOLYSINS.  109 

the  least  affected  by  our  anti-immune  body.  It  may  be  that  an  entirely 
different  receptor  apparatus  is  present  in  the  goose  and  that  this 
effects  a  combination  with  different  haptophore  groups  which  leads 
to  the  formation  of  immune  bodies  of  entirely  different  character. 

Our  further  experiments  concerned  themselves  with  the  action 
exerted  by  our  anti-immune  body  on  immune  bodies  derived  from 
rats,  guinea-pigs,  and  dogs  by  treatment  with  ox  blood.  We  found 
that  the  anti-immune  body  exerted  a  distinct  protective  action  against 
all  three  sera,  but  that  this  was  less  strong  than  that  against  the 
immune  body  of  the  rabbit.  The  protection  was  least  against  the 
serum  of  the  rat,  for  it  did  not  even  suffice  to  absolutely  protect  against 
one-half  or  one-third  of  the  fatal  dose.  Complete  solution  ensued 
in  the  presence  of  0.3  cc.  anti-immune  body  even  when  only  double 
the  usual  solvent  dose  of  immune  body  was  employed.  This  indicates 
that  this  serum  contains  a  relatively  large  amount  of  the  non-neutraliz- 
able  types  of  immune  bodies,  in  any  case  an  amount  far  greater  than 
is  contained  in  the  rabbit  serum.  In  the  guinea-pig  the  case  is  very 
similar,  the  proportion  of  double  the  solvent  doses  being  as  1:3. 
The  nearest  approach  to  the  ratio  as  found  in  the  rabbit  is  seen  in 
the  serum  of  a  dog  treated  with  ox  blood.  In  this  it  required  six 
times  the  usual  solvent  dose  to  effect  complete  solution  in  the  presence 
of  the  anti-immune  body.1 

All  this  leads  to  the  conclusion  that  in  the  immune  serum  of 
these  three  species  the  cytophile  group  of  certain  portions  is  identical 
with  the  cytophile  group  of  certain  immune  bodies  in  the  rabbit. 
Certain  particular  groups  of  the  ox  blood-cells  therefore  must  fit 
equally  into  the  receptors  of  these  different  animals.  In  view  of 
this  fact,  the  entire  absence  in  the  goat  of  that  portion  of  immune 
body  which  can  be  neutralized  by  the  anti-irnmune  body  is  of  special 
interest.  As  already  stated,  we  are  here  dealing  with  an  exception 
which  is  connected  with  the  impossibility  of  autolysin  formation. 

We  must  therefore  conclude  that  in  conformity  with  our  assump- 
tion,, the  immune  bodies  formed  in  any  single  case  by  treating  various 

1  It  is  perhaps  of  interest  to  know  that  the  immune  bodies  derived  from 
these  three  species  (guinea-pig,  rat,  and  dog)  differed  in  their  behavior  toward 
goat  blood-cells.  It  was  found  that  while  the  immune  bodies  of  guinea-pigs 
and  rats  acted  on  goat  blood,  those  of  the  dog  did  not.  This  indicates  that 
the  dog,  in  contrast  to  rabbits,  guinea-pigs,  and  rats,  possesess  no  receptors 
for  the  groups  (3  of  the  diagram,  Fig.  1)  common  to  the  blood-cells  of  oxen 
and  goats. 


110  COLLECTED  STUDIES  IN  IMMUNITY. 

animals  with  ox  blood-cells  are  not,  as  a  matter  of  fact,  of  simple 
[einheitlich]  nature.  Those  obtained  from  goats  and  geese  are  very 
markedly,  if  not  entirely,  different  from  those  of  rabbits,  while  those 
from  guinea-pigs,  rats,  and  dogs  are  partly  so. 

We  have  already  pointed  out  the  significance  of  this  circumstance 
in  §  II,  page  92.  In  all  probability  similar  conditions  obtain  for 
bacteria,  and  it  would  therefore  be  advisable  not  to  attempt  the  pro- 
duction of  bactericidal  sera  from  a  single  animal  species,  as  is  now 
customary,  but  to  make  a  preparation  containing  a  mixture  of  immune 
sera  derived  from  animals  whose  receptor  apparatus  are  as  divergent 
as  possible. 


III.    Concerning  the  Variety  of  the  Complementophile  Groups  of 
Homologous  Immune  Bodies.1 

From  the  foregoing  sections  it  will  be  seen  that  in  combating 
infectious  diseases  we  believe  it  advisable  to  employ  simultaneously 
a  great  many  bactericidal  immune  bodies  which,  in  conformity  with 
the  multiplicity  of  groups  in  the  bacterial  cell,  will  differ  in  their 
cytophile  group.  It  will  now  be  necessary  to  investigate  the  question 
of  a  difference  in  the  complementophile  groups  of  these  immune 
bodies.  However,  the  treatment  of  this  question  can  at  present  only 
be  fragmentary,  since  our  methods  in  this  field  are  still  very  incomplete 
and  definite  results  can  only  be  obtained  in  specially  favorable  cases. 

It  will  be  advisable  to  commence  this  study  with  the  immune 
serum  of  a  rabbit  treated  with  ox  blood.  In  this  it  has  already 
been  pointed  out  that  two  portions  of  immune  bodies  are  present, 
each  of  which  again  is  to  be  regarded  as  composed  of  a  number  of 
partial-immune  bodies.^  This  view  of  the  composition  of  the  im- 
mune bodies  is  supported  by  the  reactivating  experiments  in  ^-hich 
a  number  of  different  kinds  of  sera  furnished  the  complements.  This 
brings  us  to  our  present  topic. 

We  have  already  mentioned  that  the  most  favorable  results  are 
achieved  when  our  immune  body  is  activated  by  rabbit  or  guinea- 
pig  serum;  the  activation  by  means  of  goat  serum,  together  with  its 
peculiarities,  has  also  been  discussed  at  length. 

The  following  list  of  complements  shows  their  action  in  the  pres- 
ence of  varying  amounts  of  an  immune  body  from  a  rabbit  immunized 
with  ox  blood.  The  amount  of  complement  employed  was  always  ample. 

1  See  also  Ehrlich's  later  views,  page  560. 


STUDIES  OX  H.EMOLYSINS. 
TABLE  X. 


Ill 


Activating  Serum. 

Amount  of  Immune 
Body  with  which  Com- 
plete Solution  Occurs, 
cc. 

Guinea-pig  serum  
Rabbit  serum             

0.0025 
0.005 

Rat  serum       

0.005 

Goose  serum       

0  015 

Chicken  serum        

0.015 

Goat  serum         

0.05 

Pigeon  serum      

no  complement  action 

Horse  serum  *     

«            ii              it 

1  This  horse  serum,  which  had  been  freshly  obtained,  failed  also  to  reactf- 
vate  the  immune  bodies  of  a  goat  and  a  goose  which  had  been  immunized  with 
ox  blood.  Yet  it  was  not  at  all  free  from  complement,  for  even  in  amounts 
of  0.15  cc.  it  dissolved  guinea-pig  blood  completely.  It  did  not  act  on  rabbit 
blood. 

This  shows  that  when  different  sera  are  used  as  complements 
there  is  a  great  variation  in  the  amount  of  immune  body  necessary 
for  solution.  Especially  the  extreme  cases  make  it  seem  probable 
that  we  are  dealing  with  different  types  of  partial-immune  bodies, 
to  which  different  complements  in  the  serum  of  the  individual  species 
correspond.  That  the  complements  of  different  species  are  not  iden- 
tical is  admitted  even  by  Bordet,  although  he  recognizes  only  a  single 
complement  for  each  species. 

That  these  complements  are  anchored  to  the  corresponding 
immune  body  by  means  of  a  haptophore  group  may  practically  be 
regarded  as  proven,  (1)  by  our  experiments  with  blood-cells  which 
had  been  laden  with  immune  body,  and  (2)  by  the  demonstration 
of  anti-complements  which  diverted  the  complements  from  the 
immune  body. 

According  to  our  view  the  point  at  which  the  haptophore  group 
takes  hold  is  situated  in  the  complementophile  portion  of  the  immune 
body.  Hence  we  formerly  designated  the  latter  as  "  interbody  "  ; 
recently  we  term  it  "  amboceptor."  A  number  of  special  investi- 
gators have  accepted  this  view,  as  can  be  seen  from  the  designations 
used  by  them ;  thus  P.  Miiller,  "  copula  "  •  London,  "  desmon  " ; 
Metchnikoff :  "  cytase  "  =  complement ;  "  philocytase  "  =  immune  body. 

Consequently  we  arrive  at  the  view  that  in  the  mixture  of  immune 
bodies  in  the  case  under  discussion  a  number  of  different  complemen- 


112  COLLECTED  STUDIES  IX  IMMUNITY. 

tophile  groups  come  into  play.  With  the  means  at  present  at  our 
disposal  it  is  impossible,  except  in  a  few  favorable  cases,  to  deter- 
mine whether  this  plurality  of  complementophile  groups  corresponds 
exactly  to  a  like  plurality  of  cytophile  groups.  A  case  in  point 
is  that  of  the  partial  immune  body  which  is  reactivated  by  goat 
serum,  for  which  we  could  show  that  it  was  not  diverted  by  our 
anti-immune  body.1 

The  difficulty  of  a  full  analysis  of  these  cases  is  due  especially 
to  the  many  possibilities  that  must  be  considered.  It  is  possible 
that  immune  bodies  with  different  cytophile  groups  possess  the  same 
complementophile  group,  or  that  those  with  the  same  cytophile 
group  possess  different  complementophile  groups;  and  finally  it  is 
possible  that,  besides  a  particular  cytophile  group,  an  immune  body 
may  possess  two,  three,  or  more  complementophile  groups  (triceptor, 
quadriceptor) . 

In  any  case  it  may  be  considered  a  fact  that  in  the  immune-body 
mixture  different  kinds  of  complementophile  groups  come  into  play. 
Were  we  to  assume  that  the  serum  of  an  animal  species  contains 
only  a  single  complement,  we  should  have  to  regard  such  a  plurality 
of  complementophile  groups  as  evidently  a  useless  arrangement. 
It  seems  incredible  that  a  given  organism  should  form  haptophore 
groups  in  its  cells  (for  the  immune  bodies  are  merely  thrust-off  cell 
derivatives)  if  these  groups  were  never  during  life  to  come  into  action, 
but  were  only  to  be  of  service  in  case  the  organism  were  injected 
with  foreign  cells.  It  is  much  simpler  and  more  natural  to  view 
these  circumstances  from  our  standpoint,  namely,  that  the  comple- 
ments of  an  animal  are,  from  the  first,  of  manifold  variety. 

This  assumption  best  harmonizes  the  results  of  the  various  experi- 
ments which  we  have  made  from  the  beginning  of  our  studies  in 
haemolysis.  By  filtering  goat  and  horse  sera  through  Pukall  filters 
we  were  able  to  demonstrate  two  complements.  One  of  these,  fitting 


1  In  our  fourth  communication  we  have  discussed  analogous  cases  in 
detail,  subjecting  them  to  thorough  experimental  investigations.  At  that 
time,  however,  our  studies  were  directed  only  to  the  complementophile  groups. 
In  that  case  the  serum  of  guinea-pigs  immunized  with  rabbit  blood  contained 
two  immune  bodies,  of  which  one  found  its  complement  in  guinea-pig  serum 
but  not  in  rabbit  serum.  These  immune  bodies  were  present  in  the  propor- 
tion of  1 : 10.  In  another  case  mentioned  at  that  time  we  observed  consider- 
able chronological  variations  in  the  proportion  of  two  immune  bodies  with 
different  complementophile  groups. 


STUDIES  ON  H^MOLYSINS.  113 

to  an  immune  body  acting  on  rabbit  blood,  passed  through  with 
the  greatest  difficulty;  the  other,  fitting  an  immune  body  acting 
on  guinea-pig  blood,  passed  through  in  part  completely  isolated. 
We  were  further  able  to  show  that  heating  the  serum  of  a  buck 
treated  with  sheep  blood  caused  all  the  complements  excepting 
one  to  disappear.  The  one  which  withstood  the  heat  fitted  the  immune 
body  developed  by  the  immunization.  We  were  able  to  demon- 
strate the  same  thermostabile  complement  in  greater  or  smaller  amounts 
in  the  serum  of  normal  goats  and  calves.  To  again  call  attention 
to  these  experiments  is  not  superfluous,  for  Gengou  (Annal.  I'Inst. 
Pasteur,  1901)  in  spite  of  these  proofs  of  the  plurality  of  comple- 
ments, still  maintains  that  the  serum  of  each  species  contains  only 
a  single  simple  complement,  "  the  alexin." 

It  would  be  natural  to  conclude  that  there  is  a  plurality  of  com- 
plements from  the  manifold  variations  observed  in  the  comple- 
tion of  various  inactive  sera  by  normal  sera.  The  commonest, 
example  of  this,  probably  known  to  every  one  having  experience 
in  this  field,  consists  in  the  fact  that  a  certain  immune  serum  can 
be  activated  by  two  different  sera  serving  as  complement,  whereas 
other  immune  sera  can  be  activated  by  only  one  of  these  sera.  Never- 
theless from  our  standpoint  we  cannot  regard  this  method  of  proof 
as  at  all  conclusive  because  it  rests  on  the  assumption  that  for  a 
certain  species  of  blood  a  serum  contains  only  a  single  interbody 
(or  immune  body).  In  our  fourth  communication  we  have  already 
shown  that  this  assumption  does  not  hold,  even  for  the  interbodies 
of  normal  sera. 

The  assumption  of  a  plurality  of  complements  in  normal  sera  is 
supported  by  the  fact  that  by  injections  of  a  normal  serum  (which,  accord- 
ing to  our  view,  possesses  various  active  substances  which  may  be 
present  as  complements,  or,  at  times,  in  the  form  of  complementoids) 
antisera  are  formed  which  act  against  the  complements  of  various  other 
sera.  In  a  number  of  different  animals  by  injecting  various  sera  we 
have  succeeded  in  obtaining  anticomplements  acting  not  only  against 
the  serum  originally  employed,  but  also  against  certain  comple- 
ments of  rabbits  and  guinea-pigs.  According  to  Bordet's  experi- 
ments it  is  possible,  by  injecting  a  rabbit  with  guinea-pig  serum, 
to  obtain  an  isolated  anticomplement  against  a  complement  (able 
to  act  in  this  particular  case)  present  in  guinea-pig  serum.  From 
this  it  follows  that  in  these  sera,  since  they  excite  the  production 
of  different  anticomplements,  at  least  two  different  complements 


114 


COLLECTED  STUDIES  IN  IMMUNITY. 


are  concerned.  In  this  connection  it  is  particularly  interesting 
to  note  that  by  long-continued  treatment  of  a  goat  with  rabbit 
serum  we  obtained  an  anticomplement  serum  which  acted  also 
against  guinea-pig  serum.  Table  XI  will  make  this  clear.  All  of 
the  experiments  are  made  with  an  immune  body  derived  from  a 
rabbit  by  immunizing  with  ox  blood. 

TABLE  XI. 


Anticomple- 
ment Derived 
from 

Treated  with 

Protection 
against  Rabbit 
Complement. 

Protection 
against 
Guinea-pig 
Complement. 

Rabbit 

Guinea-pig  serum  

+ 

+  +  + 

Goat 

Dog  serum           

+  +  + 

+  +  + 

Goat 

Horse  serum   

+  +  + 

+  +  + 

Goat 

Rabbit  serum     

+  +  + 

+  +  + 

Rabbit 

Goat  serum  

+  + 

+  + 

Rabbit 

Sheep  serum  

+  +  + 

+  +  + 

+  +  +=  strong  protection;  ++=  fairly  strong  protection;  +  =  very  slight 
protection. 

With  the  assumption  of  a  plurality  of  complements  we  are  led  to 
the  view  that  the  various  complementophile  groups  of  the  immune  body 
here  concerned  (contained  in  rabbit  serum)  are  complemented  by  a 
like  number  of  partial  complements.  As  a  result  of  this  fact  the  possi- 
bility exists  that  certain  of  these  complements  are  not  constant,  occurring 
in  the  blood  only  from  time  to  time. 

We  may  perhaps  give  another  example  of  these  partial  com- 
plements, which  concerns  one  of  a  number  of  rabbits  treated  with 
repeated  injections  of  goat  serum.  As  already  described  in  a  previous 
communication,  this  results  in  the  disappearance  of  certain  com- 
plements and  their  replacement  by  corresponding  autoanticomple- 
ments.  In  the  example  mentioned,  this  disappearance  manifested 
itself  by  the  fact  that  large  amounts  of  the  rabbit  serum  were 
unable  to  activate  the  single  or  the  double  fatal  dose  of  the 
immune  body  from  a  rabbit  immunized  with  ox  blood.  How- 
ever, when  thirty  times  the  amount  of  immune  body  was  employed 
complete  solution  ensued.  Evidently  the  principal  portion  of  the 
complements  usually  present  had  disappeared  from  this  serum,  but  a 
partial  complement  had  remained  which  acted  on  a  partial-immune 
body  present  in  relatively  small  amounts.  The  circumstances  in  this 
case  therefore  are  entirely  analogous  to  those  above  described  in 


STUDIES  OX  H.EMOLYSIXS. 


115 


which  we  proved  that  a  particular  immune  body  present  in  small 
amounts  and  not  diverted  by  our  anti-immune  body,  finds  a  comple- 
ment in  its  own  serum  which,  in  contrast  to  the  other  complements, 
is  present  also  in  goat  serum. 

Three  things  have  thus  been  established: 

1.  Each   normal   serum   contains   a  number  of  different   com- 

plements ; 

2.  In  different  animals  a  part  of  the  complements  present  are 

either  completely  similar  or  at  least  similar  in  their  hap- 
tophore  groups; 

3.  The  immune  bodies  obtained  in  an  animal  species  represent 

a  number  of  different  complementophile  groups. 

As  a  result  of  this  demonstration  the  question  whether  or  not 
the  resultant  immune-body  mixtures  obtained  in  different  animals 
are  identical  in  their  complementophile  portion  loses  in  interest  at 
least  so  far  as  the  problems  under  discussion  are  concerned. 

Hence  we  should  merely  like  to  add  to  the  results  obtained  by 
activating  the  immune  body  of  a  rabbit  immunized  with  ox  blood, 
the  results  of  a  parallel  series  of  experiments  made  at  that  time 
with  the  same  amounts  of  reactivating  sera  but  with  the  immune  body 
from  a  goose  immunized  with  ox  blood.  (See  Table  XII.) 

TABLE  XII. 


Reactivating  Normal  Sera. 

Amount  of  the 
Rabbit  Immune 
Body  Sufficient  to 
Effect  Complete 
Solution. 
cc. 

Amount  of  the  Goose 
Immune  Body 
Sufficient  to  Effect 
Complete  Solution. 

cc. 

Guinea-pig  serum     

0  0025 

0  025 

Rabbit  serum 

0  005 

0  05 

Rat  serum       

0  005 

0  1 

Goose  serum                                  .      .  . 

0  015 

0  035 

Chicken  serum      

0  015 

0  035 

Goat  serum     

0.05 

no  "  completion" 

Pigeon  serum      

no  "completion" 

0  035 

Horse  serum                     

no  "completion" 

no  "completion" 

This  table  again  shows  that  the  Unitarian  view7,  according  to 
which  each  serum  contains  only  a  single  complement,  lacks  all  prob- 
ability, for  it  is  to  be  expected  that  in  that  case  the  zoological  rela- 
tionship of  certain  animal  groups  would  manifest  itself  in  their  com- 
plements to  a  greater  degree  than  it  actually  does.  When,  for 
example,  we  here  see  that  the  rabbit  immune  body  is  not  reactivated 


116  COLLECTED  STUDIES   IN  IMMUNITY. 

by  horse  serum  but  is  reactivated  by  goose  serum,  we  should  neces- 
sarily have  to  conclude  that  "  the  "  complement  of  the  goose  is  much 
more  closely  related  to  "  the  "  complement  of  the  rabbit  than  is 
that  of  the  horse.  From  the  Unitarian  standpoint  also  a  more 
marked  difference  should  be  manifested  by  the  complements  of 
the  goose,  the  chicken,  and  the  pigeon,  for  the  first  two  reactivate 
the  immune  body,  while  the  last  does  not.  A  priori,  therefore,  the 
Unitarian  view  is  very  improbable ;  but  aside  from  this  the  reactivat- 
ing experiment  with  the  goose  immune  body  (which  shows  this  to 
be  reactivated  by  all  three  avian  sera)  speaks  against  this  view. 

All  of  these  facts  are  readily  explained  if  we  accept  the  pluralistic 
view  that  each  serum  contains  a  large  number  of  complements,  and 
that  certain  types  have  a  wide  distribution  in  many  classes  of  animals. 
In  these  they  may  be  completely  similar,  or,  what  is  of  primary  impor- 
tance, their  haptophore  groups  may  be  identical.  It  may  very 
well  be  that  the  avian  sera  are  alike  in  the  greater  part  of  their  partial 
complements,  and  that  therefore  all  three  sera  may  in  certain  cases — 
e.g.,  with  the  immune  body  of  a  goat  immunized  with  ox  blood — reactivate 
in  like  manner.  But  it  is  not  necessary  that  these  three  species 
correspond  in  all  their  complements,  and  so  it  may  happen  that  a 
certain  partial  complement  which  is  absent  in  pigeon  serum  is  present 
in  the  other  sera.  This  occurs  in  the  above  case  with  the  immune 
body  of  the  rabbit  immunized  with  ox  blood  (and  with  that  of  the 
goat  similarly  treated). 

I  should  like  to  emphasize  one  more  point.  The  immune  body 
of  the  rabbit  immunized  with  ox  blood  is  not  reactivated  by  pigeon 
serum,  whereas  the  immune  body  of  the  goat  immunized  with  ox 
blood  is  thus  reactivated.  This  fact  in  itself  should  occasion  no  sur- 
prise whatever.  The  tissue  receptors  which  are  present  in  the  avian 
organism,  and  which  constitute  the  matrix  of  the  amboceptors  in 
question,  possess  complementophile  groups  that  fit  complements 
widely  distributed  throughout  the  avian  body.  It  is  not  at  all  remark- 
able, therefore,  that  the  immune  body  obtained  from  the  goose  finds 
complements  in  various  avian  sera.  In  like  manner  it  can  readily 
be  understood  why  pigeon  serum  is  unable  to  reactivate  the  immune 
body  of  the  rabbit  immunized  with  ox  blood. 

The  general  conclusion,  however,  that  the  avian  complements  in 
their  entirety  are  different  from  those  of  mammals,  cannot  be  drawn 
from  this,  as  is  shown  by  the  reactivation  of  the  rabbit  immune 
body  by  goose  and  chicken  sera. 


STUDIES  ON   H.EMOLYSINS.  117 

This  brief  analysis  will  show  us  that  the  complementophile  groups 
of  the  immune  bodies  do  not  in  general  possess  the  great  importance 
which  we  must  ascribe  to  the  cytophile  groups.  In  order  to  obtain 
the  greatest  therapeutic  effect  from  the  immune  bodies,  their  com- 
plementophile groups  and  the  provision  of  suitable  complements 
cannot,  of  course,  be  neglected.  In  this  connection  Donitz  (Klin. 
Jahrbuch,  1897)  first  pointed  out  the  importance,  in  the  therapy  of 
infectious  diseases,  of  finding  sufficient  sources  of  complements. 
The  conditions  determining  this  have  been  more  closely  defined  by 
Ehrlich  in  his  Croonian  Lecture  x  of  March  22,  1900,  as  can  be  seen 
from  the  following  extract: 

"Dr.  Neisser  at  the  Steglitz  Institute  sought  to  find  an  explana- 
tion of  Sobernheim's  experiments.  He  was  able  to  determine  that 
anthrax  serum  failed  in  mice  even  if  large  quantities  of  fresh  sheep- 
serum  (i.e.,  containing  an  excess  of  'complement')  were  introduced 
at  the  same  time.  The  failure  in  this  case  appears  to  be  due,  on 
the  one  hand,  to  the  destruction,  in  the  body  of  the  mouse,  of  the 
'complement'  present  in  the  sheep  serum,  and,  on  the  other  hand, 
to  the  fact  that  the  'immune  body'  yielded  by  the  sheep  does  not 
find  in  the  mouse  an  appropriate  new  'complement.' 

"From  this  it  appears  that  in  the  therapeutic  application  of 
antibacterial  sera  to  man,  therapeutic  success  is  only  to  be  attained 
if  we  use  either  a  bacteriolysin  with  a  '  complement '  which  is  stable 
in  man  (homostabile  complement),  or  at  least  a  bacteriolysin  the 
immune  body  of  which  finds  in  human  serum  an  appropriate  'com- 
plement.' The  latter  condition  will  be  the  more  readily  fulfilled 
the  nearer  the  species  employed  in  the  immunization  process  is  to 
man.  Perhaps  the  failure  which  has  as  yet  attended  the  employ- 
ment of  typhoid  and  cholera  serum  will  be  converted  into  success 
if  the  serum  be  derived  from  apes  and  not  taken  from  species  so 
distantly  removed  from  man  as  the  horse,  goat,  or  dog.  However 
this  may  be,  the  question  of  the  provision  of  the  appropriate  'com- 
plement' will  come  more  and  more  into  the  foreground,  for  it  really 
represents  the  center  round  which  the  practical  advancement  of 
the  bacterial  immunity  must  turn." 

In  view  of  the  fact  that  every  normal  serum  contains  a  great 
many  complements,  of  which  a  larger  or  smaller  part  fits  the  most 
varied  immune  bodies,  the  need  of  artificially  supplying  complements 

1  Proceedings  of  the  Royal  Society,  Vol.  66. 


118  COLLECTED  STUDIES   IN   IMMUNITY. 

would  seem  to  indicate  that  our  therapeutic  efforts  be  directed  primarily 
to  exciting  the  greatest  production  of  the  organism's  own  complements.1 
The  production  of  these  complements  can  surely  be  increased  by 
means  of  artificial  procedures ;  and  this  is  borne  out  by  a  few  experi- 
ences in  this  direction.  Thus  Nolf,  by  injecting  certain  foreign  sera, 
and  P.  Miiller,  by  injecting  pepton,  have  succeeded,  in  animal  experi- 
ments, in  increasing  the  production  of  complement.  This  increase 
may  perhaps  be  referable  to  a  hyperleucocytosis  in  accordance  with 
the  views  held  by  Metchnikoff  and  Buchner.  We  are  certain  that 
at  least  the  complements  orginally  peculiar  to  the  organism  will  be  able 
to  act  when  fitting  complementophile  groups  present  themselves ;  this 
need  not  necessarily  be  the  case,  however,  when  foreign  complements 
are  introduced.  In  this  question  it  is  of  no  consequence  whether 
the  absence  of  complement  action  is  due  to  destruction,  to  com- 
plementoid  formation,  or  to  a  combination  in  the  organism  such 
as  has  been  demonstrated  by  the  ready  binding  of  anticomple- 
ments.2  The  question  raised  by  Donitz,  relative  to  the  provision 
of  really  plentiful  sources  of  complement,  has  not  thus  far  been 
solved.  It  still  remains  to  be  seen  whether  the  interesting  in- 
vestigations of  Wassermann3  on  the  completion  of  typhoid  im- 
mune bodies  with  ox  serum  will  lead  to  results  which  can  be 
practically  utilized.  The  amount  of  complement  contained  in 
the  serum  of  the  larger  laboratory  animals  is  not,  as  a  rule,  great 
enough  to  make  the  employment  of  these  sera  applicable  for  human 
therapeutic  purposes.  Wassermann,  for  example,  found  that  with 
a  method  of  procedure  which  excluded  the  above-mentioned  diminu- 
tion of  complements  (since  he  injected  bacteria,  immune  body,  and 
complement  mixed  together  into  the  peritoneal  cavity)  it  required 
4  cc.  ox  serum  to  achieve  curative  results.  This  amount  of  serum 
in  itself  causes  severe  injury  to  the  animals  experimented  upon. 

Such  being  the  case,  it  seems  that  in  the  matter  of  supplying  com- 
plements, the    method    suggested    by   us,  namely,  the    employment    of 

1  In  his  recent   study   (Zeitschrift  fiir  Hygiene,  No.   37)    Wassermann  also 
lays  great  stress  on  the  increase  of  the  individual's  own  complements.     We 
were  especially  gratified  to  see  that  in  regard  to  the  multiplicity  of  the  comple- 
ments Wassermann  accepts  our  view  completely. 

2  This  is  also  supported   by  certain  experiments  of  von  Dungern  (Munch, 
medizin.  Wochenschrift)  concerning  the  binding  of  complement  by  certain  cells 
in  mirq. 

9  Deutsche  medizinische  Wochenschrift,  1900,  No.  18. 


STUDIES  ON   H^MOLYSINS.  *     119 

mixed  sera  which  contain  a  great  many  different  immune  bodies  would 
prove  the  most  effective;  for  with  the  multiplicity  of  the  immune  bodies 
an  increase  of  the  different  complementophile  groups  also  takes  place, 
and  thus  the  probability  increases  that  the  normal  complements  present, 
especially  those  in  the  human  organism,  can  come  into  action  most  effec- 
tively. 


IX.    CONCERNING  THE  MODE  OF  ACTION  OF  BACTERI- 
CIDAL SERA.1 

By  MAX  NEISSER,  Member  of  the  Institute,  and  Dr.  FREDERICK  WECHSBERG. 

OUR  experiences  with  diphtheria  curative  serum  have  taught  us 
that  in  antitoxin  the  employment  of  a  high  dose  of  antitoxin  is  of 
primary  importance.  It  is  immaterial  whether  an  excess  of  antitoxin 
is  administered,  since  it  may  be  regarded  as  certain  that  an  excess 
does  no  harm  and  can  on  the  contrary  only  be  of  benefit. 

Concerning  the  action  of  bactericidal  sera,  however,  the  litera- 
ture contains  a  number  of  examples  which  indicate  that  here  an  excess 
of  immune  serum  is  occasionally  injurious.  Thus  several  high 
authorities  have  published  protocols  of  therapeutic  experiments  on 
animals  which  seem  to  contain  paradoxical  results ;  for  with  the  same 
infection  and  varying  amounts  of  immune  serum  not  only  those 
animals  died  which  had  received  the  smallest  amounts  of  serum  but 
also  those  which  had  received  the  largest  amounts.  Only  those 
animals  were  protected  which  received  doses  of  immune  serum  lying 
between  these  extremes.  Such  a  protocol,  for  example,  was  published 
by  Loffler  and  Abel2  on  their  experiments  with  bacillus  coli  and  a 
corresponding  immune  serum.  Out  of  nineteen  guinea-pigs  which 
had  been  inoculated  with  the  same  amount  of  culture  (Vio  loop) 
and  had  received  varying  amounts  of  the  immune  serum,  only  six 
animals  were  protected,  those  which  had  received  doses  of  0.25  to 
0.02  cc.  Eight  animals  with  larger  doses  as  well  as  five  with  smaller 
doses  of  serum  died. 

A  similar  protocol  is  that  of  R.  Pfeiffer,3  which  states  that  of  four 
guinea-pigs  treated  with  virulent  cholera  and  a  corresponding  im- 
mune serum  only  the  two  animals  receiving  the  medium  doses 
survived. 

1  Reprinted  from  the  Munch,  med.  Wochenschr. ,  1901,  No.  18. 

2  F.  Loffler  and  R.  Abel,  Centralbl.  f.  Bact.,  1896,  Vol.  19,  page  51. 
1  R.  Pfeiffer,  Zeitschr.  f.  Hygiene,  1895,  Vol.  20,  page  215. 

120 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA.  121 

The  same  phenomenon  was  noticed  by  Leclainche  and  Morel l 
in  their  work  on  the  bacillus  of  malignant  oedema,  and  these  authors- 
had  similar  experiences  with  erysipelas  of  swine  and  with  sympto- 
matic anthrax.  As  a  result  of  this  they  concluded  that  there  was 
a  "dosis  optima  neutralisans"  of  the  immune  serum. 

Since  we  encountered  the  same  phenomenon  in  bactericidal 
test-tube  experiments  it  seemed  advisable  to  undertake  a  study  of 
these  occurrences,  especially  because  the  question  seemed  to  offer 
points  of  vantage  important  both  theoretically  and  practically. 
None  of  the  authors  above  mentioned  has  furnished  an  adequate 
explanation  of  the  phenomenon. 

In  our  experiments  the  bactericidal  action  was  determined  in  two 
ways,  namely,  with  the  aid  of  the  bioscopic  method  previously 
described  by  us,2  and  by  means  of  plate  countings.  The  methods 
gave  identical  results  even  in  parallel  series.  In  order,  therefore, 
to  facilitate  looking  over  the  results  we  shall  here  give  only  the 
results  obtained  by  the  counting  method. 

The  method  of  procedure  was  generally  as  follows:  Vsooo  cc.  of  a 
one-day  bouillon  culture  of  the  bacterium  hi  question  was  put  into 
each  of  a  series  of  test-tubes.  To  this  were  added  varying  amounts 
of  immune  serum  inactivated  at  56°  C.  and  equal  amounts  of  the 
complementing  active  serum;  or  in  another  series,  equal  amounts 
of  immune  serum  and  varying  amounts  of  the  complementing  serum. 
It  was  so  arranged  that  all  the  tubes  contained  equal  emounts  of 
fluid,  usually  2.5  cc.  Dilutions  were  made  with  0.85%  salt  solution. 
Furthermore  three  drops  of  bouillon  were  added  to  each  tube,  for 
we  had  convinced  ourselves  that  this  assured  a  good  growth  in  the 
control  tubes.  Numerous  control  tests  were  necessary  nevertheless, 
even  if  only  to  test  the  sterility  of  the  sera  employed.  The  specimens 
were  kept  at  37°  C.  for  three  hours  and  then  plated  on  agar,  using 
five  drops  from  pipettes  of  uniform  size  for  each  plate.  The  results 
were  noted  by  comparison  and  estimation,  somewhat  after  the 
following  scheme:  0,  isolated,  hundreds,  thousands,  infinite  number. 

Omitting  the  very  extensive  preliminary  tests  the  following 
example  is  given  to  show  the  phenomenon  studied  by  us.  The 
immune  serum  employed  was  obtained  from  a  rabbit  by  treatment 


1  Leclainche  and  Morel,  La  Serotherapie  de  la  septicemie  gangreneuse,  AnnaL 
de  1'Inst.  Pasteur,  1901,  Xo.  1. 

3  Munch,  med.  Wochenschr.,  1900,  Xo.  37. 


122 


COLLECTED  STUDIES   IN  IMMUNITY. 


with  vibrio  Metchnikoff.  This  serum  was  inactivated  by  heating 
to  57°  C.  for  half  an  hour.  Normal  active  rabbit  serum  served  as 
complement. 

TABLE  I. 


Inactive  Im- 

Amount 

mune  Rabbit 

Normal  Active 

Number  of 

of 
Culture. 

Serum  against 
Vibrio 

Rabbit  Serum 
as  Complement. 

Colonies  on  the 
Plate. 

Metchnikoff. 

cc. 

cc. 

^ 

1.0 

0.3 

00 

H3        j£ 

0.5 

0.3 

00 

III 

0.25 
0.1 

0.3 
0.3 

Many  thousands 
Several  hundred 

03*3  -^ 

0.05 

0.3 

About  100 

«n  w  t>  • 

0.025 

0.3 

About  50 

°  c^ 

0.01 

0.3 

0 

WJ3   O 

0.005 

0.3 

0 

°'3^Q 

0.0025 

0.3 

About  100 

joJa*  £ 

0.001 

0.3 

00 

Tj 

0.0005 

0.3 

00 

Control      I      .... 



— 

00 

II 

0.01 



00 

"       III 

1.0 



o 

"        IV  

0.3 

00 

"         V  



1.0 

0 

Three  drops  of  bouillon  to  each  tube.  All  the  tubes  filled  to  the  same  volume 
with  0.85%  salt  solution,  then  placed  into  the  thermostat  at  37°  C.  for  three 
hours.  Finally,  five  drops  of  each  plated  on  agar. 

This  experiment  shows  that  the  inactive  immune  serum  alone 
is  innocuous  to  vibrio  Metchnikoff  (Control  II);  also  that  0.3  cc.  of 
the  active  normal  rabbit  serum  alone  is  innocuous.  However  when, 
for  example,  0.01  cc.  immune  serum  is  mixed  with  0.3  cc.  normal 
active  rabbit  serum,  the  many  thousand  germs  inoculated  are  killed. 
In  the  same  way  0.005  cc.  immune  serum  plus  0.3  cc.  normal  active 
rabbit  serum  also  causes  the  death  of  all  the  organisms.  With 
smaller  amounts  of  immune  serum  (but  with  the  same  amount  of 
the  complementing  serum  as  before)  the  destruction  of  the  germs  is 
incomplete,  while  with  still  smaller  amounts  there  is  no  destruction 
whatever.  But  the  destructive  effect  also  becomes  less  when  more  than 
0.01  cc.  immune  serum  is  used,  so  that  with  0.5  cc.  immune  serum 
no  destructive  at  all  can  be  observed.  Hence  if  we  had  tested  only 
the  mixture  of  0.5  cc.  of  this  immune  serum  plus  0.3  cc.  normal 
active  rabbit  serum  we  should  certainly  not  have  supposed  that 
^we  were  dealing  with  a  powerful  immune  serum.  That  this  action 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA 


123 


is  due  only  to  the  serum's  content  of  immune  body  is  shown  by  the 
following  experiment  hi  which  inactive  immune  serum  is  compared 
with  inactive  normal  serum  of  the  same  species,  both  sera  being 
complemented  with  active  normal  serum. 


TABLE  II. 


Amount  of  Culture. 

Amount  of 
the  Com- 
plementing 
Normal, 
Active 
Rabbit- 
serum, 
cc. 

Number  of  Colonies  on  a  Plate  on  the  Addition 
of  Serum  from  a  Rabbit  Immunized 
against  Vibrio  Metchnikoff,  the  Serum 
having  been  Inactivated. 

— 

1.0  cc. 

ice. 

ACC. 

Aec. 

-^Vtf  cc-  °f  a  one-day  bouil- 
lon culture  of  vibrio 
Metchnikoff  

r   1-° 
j 

i  _ 

00 
00 

00 
00 
00 

a  few 
many 
thousands 

00 

0 
0 

00 

0 
0 

00 

Amount  of  Culture. 

Amount  of 
Normal 
Active 
Rabbit- 
serum, 
cc. 

Number  of  Colonies  on  a  Plate  on  the  Addition 
of  Inactive  Normal  Rabbit-serum. 

Ice. 

ice. 

Ace. 

Ace. 

T^U  cc.  of  a  one-day  bouil- 
lon culture  of  vibrio 
Aletchnikoff  

!J° 

Oo 
00 

oo 

00 
00 
00 

00 
00 
00 

00 
00 

00 

Control     I.  ^nnr  cc-   bouillon  culture  4-2  cc.  0.85%  salt  sol. +3  drops  of 

bouillon,  planted  as  above,  result  oo. 
"         II.  Sterility  of  the  immune  serum,  0. 
"       III.        "        "    '.'    inactive  normal  rabbit-serum,  0. 
"        IV.        "        "    <t   active  normal  rabbit-serum,  0. 
All  the  tubes  made  up  to  equal  volumes  with  0.85%  salt  solution,  then 
placed  into  a  thermostat  at  37°  C.  for  three  hours.     Finally,  five  drops  of  each 
specimen  plated  on  agar. 

This  experiment,  too,  shows  that  Vie  cc.  immune  serum  plus 
1  cc.  or  1/3  cc.  normal  active  rabbit  serum  kills  the  germs  completely; 
while  larger  doses  of  the  immune  serum  are  less  effective.  The  addi- 
tion of  normal  inactive  rabbit  serum  has  no  effect. 

The  same  phenomenon  can  be  demonstrated  in  another  man- 
ner. For  the  complementing  serum  any  active  serum  is  used  which 
by  itself  already  possesses  a  slight  destructive  action.  If  to  such  a 
serum  varying  amounts  of  an  inactive  immune  serum  are  added, 
it  will  at  times  be  found  that  small  quantities  of  the  latter  increase 


124 


COLLECTED  STUDIES  IN  IMMUNITY. 


the  action  of  the  normal  active  serum,  while  somewhat  larger 'quan- 
tities weaken  the  action.  Still  larger  quantities  may  inhibit  the 
action  completely. 

In  the  following  experiment  an  immune  serum  was  employed 
which  had  been  obtained  by  immunizing  a  goat  with  vibrio  Nord- 
hafen.  This  serum  was  inactivated  by  heating  it  to  57°  C.  Normal 
active  goat  serum  served  as  complement.  (See  Table  III.) 

The  first  column  shows  that  normal  active  goat  serum  by  itself 
kills  bacteria,  even  in  doses  of  about  0.1  cc.  The  fourth  and  fifth 
columns  show  that  this  bacteriolytic  effect  of  the  normal  active  goat- 
serum  is  in  no  way  affected  by  the  addition  of  1.0  cc.  or  0.1  cc.  in- 
active normal  goat  serum.  From  the  third  column  we  see,  however, 
that  if  we  add  to  the  normal  active  goat-serum  0.1  cc.  inactive  im- 
mune serum,  the  bacteriolytic  effect  of  the  former  is  lowered,  and 
that  it  is  almost  neutralized  when  1.0  cc.  of  the  inactive  immune 
serum  is  added.  (Column  2.) 


TABLE  III. 


1 

2 

3 

4 

5 

Amount  of 
Culture. 

of  Com- 
plement- 
ing Nor- 
mal Active 
Goat 

Number  of  Colonies  on  a  Plate  on  Addition  of 
Inactive  Goat  Immune  Serum  against 
Vibrio  Nordhafen. 

Number  of  Colonies 
on  a  Plate  on 
Addition  of  Inac- 
tive Normal 

Serum. 

Goat  Serum. 

cc. 

— 

1.0  cc. 

0.1  cc. 

1.0  cc. 

0.1  cc. 

Tfff  CC.  of  a 

f  1.0 

0 

about  50 

0 

0 

0 

one-day 

0.5 

0 

many  hundreds 

0 

0 

0 

bouillon 

0.25 

0 

oo 

0 

0 

0 

culture 

\   0.1 

0 

00 

several  hundred 

0 

0 

of  vibrio 

0.05 

about  50 

00 

00 

about  10 

a  few 

Nord- 

0.025 

00 

00 

00 

00 

00 

hafen. 

^— 

~~     ~ 

00 

00 

00 

00 

Control     I.  yfo  cc.  bouillon  culture +  2  cc.  0.85%  salt  solution  +  3  drops 
bouillon  ==  oo . 

"         II.  Sterility  of  the  inactive  immune  serum,  0. 

"       III.         "        "    "         "        normal  goat  serum,  0. 

"        IV.        "        "    "    active  normal  goat  serum,  0. 
Three  drops  of  bouillon  to  each  tube. 

All  the  tubes  made  up  to  equal  volumes  with  0.85  %  salt  solution. 
Kept  in  the  thermostat  at  37°  C.  for  three  hours. 
Finally,  two  drops  of  each  specimen  plated  on  agar. 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA.  125 

The  same  phenomenon  is  shown  by  the  following  protocol: 

TABLE  IV. 


Amount  of 

the  Comple- 

Number of  Colonies  on  a  Plate  on  the  Addition  of  Inactive 

Amount 

menting 
Active 

Goat  Immune  Serum  directed  against  Vibrio  Nordhafen. 

of 

Normal 

Culture. 

Guinea-pig 
Serum. 

— 

1.0  cc. 

0.1  cc. 

0.01  cc. 

cc. 

1.0 

0 

many  thou- 
sands 

a  few 

0 

0.5 

0 

almost  oo 

about  100 

0 

y^5  cc.  of  a 
one-day 

0.25 

a  few 

oo 

several  hun- 
dred 

a  few 

bouillon 
culture 

0.1 

several  thou- 

i 

00 

oo 

about  100 

of  vibrio 
Xord- 
hafen. 

0.05 
0.025 

00 
00 

00 
00 

00 
00 

many  hun- 
dred 

00 

• 

00 

CO 

00 

Amount  of 

Amount  of  Culture. 

the  Comple- 
menting 
Active  Nor- 
mal Guinea- 

Number  of  Colonies  on  a  plate  on  the  Addition  of 
Inactive  Normal  Goat  Serum. 

pig  Serum. 

cc. 

1.0  cc. 

0.1  cc. 

0.01  cc. 

1.0 

0 

0 

0 

0.5 

about  100 

0 

0 

yfo  cc.  of  a  one- 
d  a  y  bouillon 
culture  of  vibrio 
Xordhafen 

0.25 
0.1 
0.05 
0.025 

a  few  hundred 

00 
00 
00 

a  few 
a  few  hundred 

00 
00 

a  few 
several  thous. 

00 
00 

1 

00 

00 

00 

Control      I.  ifo  cc.  bouillon  culture +  2  cc.  0.85%  salt  solution +  3  drops 

bouillon.     Result,  oo. 

"         II.  Sterility  of  the  goat  immune  serum,  0. 
' '       III.         "        "    "    normal  goat  serum,  0. 
"        IV.        "        "    "        "       guinea-pig   serum,  0.      Three    drops  of 

bouillon  to  each  tube. 

All  the  tubes  made  up  to  an  equal  volume  with  0.85%  salt  solution. 
Kept  in  the  thermostat  at  37°  C.  for  three  hours.     Finally,  two  drops  of 
each  plated  on  agar. 

We  succeeded  in  obtaining  similar  results  in  such  experiments 
with  the  following  combinations : 1 

1  We  should  like  to  call  attention  to  a  case  which  we  encountered  a  number 
of  times.     We  found  that  an  immune  serum  obtained  from  a  goat  could  be 


126  COLLECTED  STUDIES  IN  IMMUNITY. 

Typhoid  +  inactive  immune  serum  (dog)  +  normal  active  guinea-pig  serum. 

Vibrio  Nordhafen  +  inactive  immune  serum  (rabbit)  +  normal  active  horse 

serum; 

"  "  "  "  "  "        +  normal    active   goat 

u  tt  n  «  tt  it  serum; 

"  "  "  "  "  "  +  normal  active  sheep 

serum; 

"  "  "  "  "  "  +  normal  active  guinea- 

pig  serum. 

In  order  to  meet  the  objection  that  the  agglutinins  may  possibly 
have  interfered  in  the  experiments  we  have  devised  the  following 
method  of  demonstrating  the  phenomenon  in  question: 

Typhoid  bacilli  were  subjected  for  one  hour  at  37°  C  to  the  action 
of  inactive  immune  serum  derived  from  a  dog.  As  we  know  from  the 
hsemolytic  experiments  of  Ehrlich  and  Morgenroth,  this  results  in 
anchoring  the  interbody  present  in  the  immune  serum  to  the  bacteria. 
The  mixture  was  then  centrifuged  and  the  fluid  poured  off.  After  care- 
fully shaking  the  sediment  with  a  little  fluid.it  was  divided  into  two 
equal  parts,  to  one  of  which  inactive  immune  serum  (dog)  was  added, 
while  the  other  received  some  normal  inactive  dog-serum.  Finally 
there  was  added  to  both  portions  the  same  amount  of  a  complement- 
ing serum  (normal  active  guinea-pig  serum)  which  by  itself  was  able 
to  kill  the  bacteria.  At  the  end  of  three  hours  plate  cultures  were 
made  in  the  usual  manner.  The  results  showed  that  no  destruction 
had  occurred  in  the  tube  containing  the  excess  of  immune  serum, 
whereas  the  culture  in  the  other  tube  had  been  killed. 


reactivated  for  Vibrio  Nordhafen  by  a  complement  derived  from  rabbits.  In 
this  combination  we  again  observed  the  phenomenon  of  deflection  of  com- 
plement by  an  excess  of  immune  body.  But  even  normal  inactivated  goat 
serum  (which  contains  interbody)  when  used  in  exactly  the  same  amounts 
manifested  deflection  of  complement.  Since  no  quantitative  difference  could 
be  discovered  between  the  immune  serum  and  the  normal  serum,  we  assume 
that  in  this  case  the  deflection  of  complement  has  been  effected  by  a  substance 
in  normal  goat  serum,  as,  for  instance,  another  interbody  of  special  affinity 
or  perhaps  a  normal  anticomplement.  Not  every  complement  can  be  used 
to  reactivate  a  serum,  for  the  complement  may  be  deflected  from  the  place  of 
its  intended  action  by  any  interbody,  provided  merely  that  this  possesses 
sufficient  affinity  for  the  complement.  It  will  be  necessary  to  seek  experi- 
mentally for  combinations  in  which  such  disturbing  deflections  are  absent  and 
in  which  the  difference  in  the  affinity  of  the  interbody  which  may  be  normally 
present  and  of  that  produced  artificially  in  large  quantities  becomes  very  mani- 
fest. 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA.  127 

All  these  experiments  show  that  the  effect  produced  by  a  given  amount 
of  complementing  serum,  just  sufficient  to  reactivate  a  definite  quantity 
of  inactive  immune  serum,  was  diminished  when  large  amounts  of 
immune  serum  were  employed.  In  like  manner  it  was  possible  to  in- 
hibit the  activity  of  a  normal  serum  which  was  bactericidal  by  itself, 
by  the  addition  of  large  amounts  of  the  immune  serum. 

It  seems  to  us  that  an  explanation  of  these  important  phenomena 
is  possible  only  on  the  basis  of  the  newer  views  of  Ehrlich  and  Mor- 
genroth.  From  the  work  of  these  authors  on  haemolysins  and  from 
our  own  bacteriolytic  experiments  we  know  that  the  immune  serum 
contains  a  thermostable  interbody  (amboceptor)  which  while  itself 
inactive  renders  the  complement  effective  by  linking  itself,  on  the 
one  hand,  to  the  bacterium  or  erythrocyte  to  be  dissolved,  and  on  the 
other  to  the  complement.  The  complements,  as  is  well  known,  are 
thermolabile  and  are  contained  in  normal  sera.  But  the  interbody 
may  also  be  normally  present  in  a  serum.  This  follows  from  the 
side-chain  theory,  and  has  already  been  emphasized.1  An  instance 
ef  this  is  shown  in  Table  IV.  The  normal  active  guinea-pig  serum 
contained  complement  and  interbody.  But  besides  this  it  contained 
additional  complement,  which  became  manifest  when  more  inter- 
body, in  the  form  of  inactive  immune  serum,  was  added.  In  example 
II  it  was  impossible  to  demonstrate  an  interbody  hi  the  normal 
serum,  for  this  by  itself  did  not  kill  the  bacteria  even  though  inactive 
normal  serum  was  added.  It  did,  however,  contain  complement, 
and  this  became  manifest  when  inactive  immune  serum  was  added. 
These  phenomena  are  exactly  like  those  observed  with  haemolysins 
which  have  recently  been  so  carefully  studied.  This  one  phenomenon, 
however,  of  the  ineffectiveness  of  large  doses  of  immune  serum  has 
not  thus  far  been  encountered  in  haemolysins.  This  is  apparently 
due  to  differences  in  the  affinities  of  the  interbodies,  as  we  shall  pres- 
ently show. 

In  Fig.  1,  on  the  next  page,  All  represents  schematically  a  bac- 
terium a  with  a  number  of  receptors;  for  there  are  many  reasons 
why  we  should  assume  that  each  bacterium  possesses  a  number  of 
receptors  of  the  same  kind.  According  to  the  side-chain  theory, 
if  we  inject  this  bacterium  into  an  animal  an  overproduction  of  the 
corresponding  group  will  occur,  resulting  in  a  serum  which  is  rich 
in  body  b.  This  body  6,  however,  is  not  able  by  itself  to  injure  the 

1  Deutsche  med.  Wochenschr.,  1900,  No.  49. 


128 


COLLECTED  STUDIES  IN  IMMUNITY. 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA.  129 

bacteria,  and  a  bacterium  all  of  whose  receptors  are  laden  with 
b  need  not  at  all  be  injured  in  its  vitality.  Body  b  normally  possesses 
a  peculiar  function,  namely,  to  serve  as  a  coupling  member  or  link, 
and  hence  it  possesses  two  groups  (amboceptor).  In  this  particular 
case  one  of  these  groups  fits  the  receptor  of  the  bacterium,  the  other 
possesses  a  peculiar  relation  to  those  normal  ferment-like  constitu- 
ents of  sera  which  Ehrlich  has  termed  complements.  When  there- 
fore to  a  normal  serum  which  contains  suitable  complement  we 
add  equivalent  amounts  of  immune  serum,  the  condition  pictured 
in  A  I  will  result.  On  adding  the  corresponding  bacterium  to  this 
we  get  the  condition  shown  in  A  II,  in  which  all  the  bacterial  receptors 
are  occupied  with  immune  bodies,  or,  more  accurately,  with  immune 
bodies  which  on  their  part  are  loaded  with  bacteriolytic  comple- 
ment c.  In  the  case  here  presented  we  shall  say  that  it  requires 
the  occupation  of  all  the  receptors  with  complemented  interbodies 
to  cause  the  death  of  the  bacterium. 

If  now  to  an  equivalent  mixture  of  complement  and  interbody 
we  add  an  excess  of  interbody,  it  will  be  possible  for  only  a  part 
of  the  interbody  to  be  loaded  with  complement,  leaving  a  portion 
of  the  interbody  uncomplemented.  On  adding  the  corresponding 
bacteria  a  number  of  conditions  may  result ;  the  affinity  of  the  inter- 
body for  the  bacterial  receptor  may,  as  a  result  of  the  loading  with 
complement,  (1)  remain  unchanged,  (2)  it  may  thereby  be  increased, 
or  (3)  be  diminished. 

In  the  figure,  B  II  shows  the  condition  of  increased  affinity. 
Of  the  six  interbodies  only  those  combine  with  the  bacterium  which 
have  become  laden  with  complement.  In  this  case  therefore  the 
excess  of  interbodies  will  have  no  influence  on  the  bactericidal  effect. 
The  condition  is  really  the  same  as  A  II,  except  that  free  interbody 
is  also  present. 

C  II  shows  the  condition  of  unchanged  affinity.  In  this  case, 
if  we  add  the  bacterium  to  the  mixture  of  complement  and  excess 
of  interbody,  all  the  receptors  of  the  bacterium  will,  to  be  sure,  be 
occupied  by  interbodies,  but  this  will  be  entirely  without  any  regard 
to  the  fact  that  these  interbodies  are  or  are  not  loaded  with  com- 
plement. It  may  therefore  happen  that  only  a  few  of  the  bacterial 
receptors  will  be  occupied  by  complemented  (i.e.  active)  interbodies, 
while  the  rest  of  the  bacterial  receptors  are  occupied  by  uncom- 
plemented (hence  inactive)  interbodies.  As  already  mentioned, 
however,  the  vitality  of  such  a  bacterium  is  not  necessarily  destroyed. 


130  COLLECTED  STUDIES  IN  IMMUNITY. 

D II  represents  the  last  conceivable  case.  It  is  assumed  that 
the  "  completion  "  of  the  interbody  has  resulted  in  a  diminution 
of  the  latter's  affinity  for  the  bacterial  receptor.  In  this  case  pri- 
marily only  the  uncomplemented  interbodies  will  combine  with 
the  bacterial  receptors,  while  the  free  fluid  will  contain  complemented 
interbodies. 

In  cases  C  II  and  D  II,  therefore,  the  excess  of  interbody  is  not 
without  important  results;  for  whereas  in  mixtures  of  equivalent 
amounts  of  complement  and  interbody  all  the  interbodies  are  com- 
plemented and  so  made  active,  the  excess  of  interbody  will  exert 
a  deflecting  action  on  the  complements  in  case  C  II  as  well  as  in  D  II, 
thus  diminishing  the  end  results. 

The  conditions  shown  in  B  II  are  apparently  those  which  apply 
to  the  hsemolysins,  for  extensive  investigations  in  this  direction  by 
Ehrlich  and  Morgenroth,  concerning  which  we  are  permitted  to 
report,  have  shown  that  deflection  of  complement  by  an  excess  of 
interbody  is  not  observed  in  haemolysis.  In  this  case  only  the 
complemented  interbodies  seem  primarily  to  be  anchored  by  the 
receptors  of  the  erythrocytes. 

In  the  bactericidal  sera  investigated  by  us,  however,  the  deflec- 
tion of  complement  shown  in  C II  and  D  II  is  observed,  though 
of  course  we  are  as  yet  unable  to  say  which  of  the  two  possible  modes 
is  present  in  any  particular  case.  The  same  explanation  which 
we  have  given  for  the  phenomenon  observed  in  vitro  must  a.lso  be 
held  to  apply  to  the  experiments  on  animals,  at  least  so  far  as  the 
phenomenon  above  described  was  observed.  It  is  perfectly  obvious 
that  when  appropriate  affinities  of  the  interbody  exist  and  when 
there  is  a  marked  disproportion  between  complement  and  inter- 
body, a  deflection  of  complement  by  the  excess  of  interbody  can 
occur  in  the  animal  body. 

The  phenomenon  of  deflection  as  described  may  perhaps  present 
further  points  for  study.  We  know  that  immunization  causes  an 
increase  only  of  the  interbody  and  that  therefore  every  immune 
serum  presents  a  deficiency  of  complement  in  comparison  to  inter- 
body. Hence  it  is  conceivable  in  a  highly  immune  animal,  i.e.  one 
in  which  through  immunization  a  great  increase  of  interbody  has 
occurred,  that  after  infection  the  phenomenon  of  complement  deflec- 
tion through  the  excess  of  immune  body  could  occur.  That  it  actu- 
ally does  occur  we  conclude  from  the  following  statement  by  R. 
Pfeiffer: 


MODE  OF  ACTION  OF  BACTERICIDAL  SERA.  131 

"  It  has  frequently  happened  to  me  that  highly  immunized  guinea- 
pigs  died  after  an  injection  of  moderate  amounts  of  virus.  On 
section  there  were  then  found  in  the  peritoneum  living  vibrios,  some- 
times even  in  considerable  numbers.  Notwithstanding  this  the 
heart  blood  of  the  cadaver  when  introduced  into  new  guinea-pigs 
manifested  the  strongest  power  to  dissolve  vibrios." 

It  is  therefore  conceivable  that  an  individual  can  lose  its  natural 
resistance  by  producing  too  large  an  amount'  of  interbody  in  pro- 
portion to  the  amount  of  its  complement.  Such  an  excess  of  inter- 
body  then  would  act  injuriously  rather  than  helpfully. 

This  phenomenon  is  also  of  some  theoretical  significance.  While 
it  can  readily  be  explained  by  means  of  the  views  of  Ehrlich  and 
Morgenroth,  it  appears,  to  us  at  least,  to  be  absolutely  irreconcilable 
with  the  theory  of  Bordet.  This  author,  as  is  well  known,  regards 
Ehrlich's  interbody  as  a  substance  capable  of  sensitizing  the  bac- 
teria whereby  they  are  made  vulnerable  to  the  action  of  the  solvent 
"  alexin  "  (Ehrlich's  complement).  If  this  were  the  case  it  would 
be  absolutely  incomprehensible  how  an  excess  of  sensitizing  sub- 
stance could  diminish  the  total  effect;  at  the  most  such  an  excess 
could  only  increase  the  sensitizing  action,  not  decrease  it.  Since, 
however,  we  have  actually  observed  this  decrease  very  frequently, 
we  must  regard  this  as  a  weighty  objection  to  Bordet's  theory. 

Equally  incomprehensible  from  Bordet's  standpoint  is  the  fol- 
lowing observation.  As  has  been  shown  above  it  is  possible  to  over- 
come the  action  of  an  equivalent  mixture  of  interbody  and  com- 
plement (the  mixture  itself  being  fatal)  by  adding  a  large  excess 
of  interbody  to  it.  When,  however,  through  the  addition  of  more 
complement  the  equivalence  of  the  mixture  is  again  restored,  the 
action  returns.  This  action  therefore  depends  not  only  on  the  abso- 
lute amounts  of  complement  and  interbody,  but  also  and  essentially 
on  the  proportion  in  which  these  two  substances  are  present.  This 
is  true  at  least  in  the  sense  that  not  very  much  more  interbody 
should  be  present  than  is  required. 


X.    THE  DEFLECTION  OF  COMPLEMENTS  IN  BACTERI- 
CIDAL TEST-TUBE  EXPERIMENTS.1 

By  Dr.  A.  LIPSTEIN,  Assistant  in  the  Bacteriological  Division. 

IN  a  study  published  in  190 1,2  Neisser  and  Wechsberg  described 
a  peculiar  phenomenon  occurring  hi  bactericidal  test-tube  experi- 
ments. This  phenomenon  consisted  in  the  fact  that  the  bacteria 
were  not  killed  despite  the  presence  of  the  appropriate  bacterial 
amboceptor  (immune  body)  and  complements  when  a  compara- 
tively large  excess  of  amboceptor  was  present.  This  fact,  for  which 
all  other  explanations  failed,  was  explained  by  the  authors  on  the 
basis  of  Ehrlich  and  Morgenroth's  views.  They  assumed  that,  with 
certain  conditions  of  affinity,  an  excess  of  amboceptors  exerts  a 
deflecting  and  at  the  same  time  a  diluting  action  on  the  complement; 
as  a  result  the  complement  does  not  combine  with  the  amboceptors 
anchored  to  the  bacteria,  but  with  the  superfluous  free  amboceptors, 
while  the  amboceptors  which  are  anchored  to  the  cells  remain  without 
any  complement.  Now  since  only  those  complements  exert  a  bac- 
tericidal action  which  are  anchored  to  the  bacteria  by  means  of  the 
amboceptors,  it  follows  that  in  this  case  there  will  be  no  bactericidal 
action.  Naturally  this  phenomenon  of  deflection  of  complement 
does  not  occur  with  every  combination  of  amboceptor  and  comple- 
ment, but  only  when  certain  conditions  of  affinity  are  present.  Later 
I  shall  be  able  to  show  how  the  same  amboceptor  in  excess  exerts  a 
deflecting  action  on  one  complement  while  it  fails  to  do  so  on  two 
other  complements.  Because  of  the  theoretical  importance  of  this 
phenomenon  and  its  explanation,  a  continuation  of  the  experiments 
of  Neisser  and  Wechsberg,  taking  special  cognizance  of  the  objections 
since  made,  seemed  desirable. 

1  Reprinted  from  Centralblatt  fur  Bact.,  Vol.  XXXI,  No.  10,  1902. 

2  See  page  120  of  this  volume;  also  Wechsberg,  Zeitschr.  f.  Hygiene,  Vol.  39, 
1902. 

132 


BACTERICIDAL  TEST-TUBE  EXPERIMENTS. 


133 


In  the  following  experiments  the  method  is  the  same  as  that  of 
the  above  authors,  to  whose  work  I  refer  for  these  details.  The 
phenomenon  of  the  deflection  of  complement  can  be  exhibited  in 
two  ways:  First  by  employing  as  a  source  of  complement  an  active, 
in  itself  not  bactericidal  serum  and  showing  that  when  decreasing 
amounts  of  inactive  immune  serum  are  added,  only  the  medium 
amounts  of  the  same  exert  a  bactericidal  effect,  whereas  both  the 
larger  and  the  smaller  amounts  are  ineffective.  The  results  shown 
in  Table  I  will  serve  as  an  example  of  this. 

TABLE  I.1 


Amount  of  Culture. 

Amount 
of  the 
Comple- 
menting 
Active 
Pigeon 
Serum. 

Number  of  Colonies  on  a  Plate  on  the  Addition  of 
Inactive  Chicken  Immune  Serum  Directed  against 
Vibrio  Metchnikoff. 

1.0 
cc. 

0.3 
cc. 

0.1 
cc. 

0.03 
cc. 

0.01 
cc. 

0.003 
cc. 

0.001 
cc. 

0.0003 
cc. 

0.0001 
cc. 

t  Jff  cc.  of  a  one-day  } 
bouillon  culture  of  (• 
vibrio  Metchnikoff  j 

0.4 

oo 

00 

00 

20 
to 
30 

0 

0 

0 

many 
thou- 
sand 

00 

Control      I.  -glis  cc.  bouillon  culture +  2  cc.  0.85%  salt  solution.     Result,  oo. 
II.  0.4  cc.  active  pigeon  serum  +-^  cc.  bouillon  culture.    Result,  oo 
"       III.  Sterility  of  all  the  sera,  0. 

The  second  method  consists  in  employing  a  serum  or  serum  mix- 
ture which  will  kill  the  amount  of  bacteria  employed.  By  adding 
to  this  decreasing  amounts  of  inactive  immune  serum  (or,  as  a  con- 
trol, inactive  normal  serum)  it  is  found  that  the  immune  serum, 
in  proportion  to  the  amount  added,  exerts  an  antibactericidol  effect, 
whereas  the  normal  serum  fails  entirely  to  do  this  or  does  so  in  a 
very  much  less  degree.  This  is  illustrated  by  the  results  in  Tables 
III  and  IV,  columns  1  and  2. 

In  opposition  to  the  explanation  furnished  by  Neisser  and 
Wechsberg,  according  to  which  the  deflection  of  complements  is 
caused  by  an  excess  of  amboceptors,  the  following  points  have  been 
raised : 

A.  The  phenomenon  is   due  to  agglutination   of  the  bacterial 

culture  ; 

B.  It  is  due  to  normal  anticomplements  (Metchnikoff). 


1  Each  tube  also  receives  three  drops  of  buillon  as  in  Xeisser  and  Wechs- 
berg's  exepriments.     This  applies  also  to  the  rest  of  our  experiments. 


134  COLLECTED  STUDIES  IN  IMMUNITY. 

C.  It  is  due  to  anticomplements  which  arise  during  the  immu- 
nizing process  (Gruber). 

I   shall   critically   examine   each  of  these   objections   beginning 
with  the  first. 


A.    Is  the  Deflection  of  Complements  in  any  way  Connected  with 

Agglutination  ? 

This  very  natural  objection,  namely  that  each  immune  serum 
agglutinates  the  corresponding  bacteria  and  that  it  is  this  mechanical 
effect  of  clumping  which  causes  the  bactericidal  power  of  the  immune 
serum  to  fail,  Neisser  and  Wechsberg  sought  to  overcome  by  the 
following  method  of  procedure.  They  first  agglutinated  the  bacteria 
which  were  to  be  used  in  the  cultures,  and  then  studied  the  effect 
that  a  normal  and  an  immune  serum  exerted  on  these,  with  the 
result  that  a  deflection  of  complement  was  obtained  only  with  the 
immune  serum.  The  following  experiment  also  shows  that  the 
agglutinating  action  of  an  immune  serum  is  in  no  way  the  cause 
of  the  deflection  of  complement;  for  in  it  I  succeed  in  showing 
that  an  immune  serum  which  agglutinates  strongly  is  nevertheless 
unable  to  exert  any  deflecting  action  on  the  complements. 

In  this  experiment  two  immune  sera  acting  against  vibrio 
Metchnikoff  are  employed,  namely,  that  of  a  goose  (A)  and  that 
of  a  goat  (3).  Both  sera  strongly  agglutinate  vibrio  Metchnikoff, 
i.e.,  even  in  a  dilution  of  1 : 1000.  The  method  of  procedure  is  such 
that  decreasing  amounts  of  the  inactive  sera  were  reactivated  with 
rabbit  serum  (column  1)  and  with  pigeon  serum  (column  2).  Now 
while  the  immune  serum  of  the  goat  (B)  shows  a  typical  picture  of 
deflection  of  complement,  the  immune  serum  of  the  goose  (A),  whose 
bactericidal  power  is  just  as  strong  as  that  of  the  goat  serum,  is 
unable  despite  this  large  content  of  amboceptor  to  deflect  the  com- 
plement. This  proves  that  the  agglutinating  action  and  that  of 
complement  deflection  are  two  properties  of  one  and  the  same  immune 
serum,  which  may  exist  side  by  side,  but  that  agglutination  in  no 
way  causes  deflection  of  complement. 

According  to  our  view  the  reason  why  the  surplus  amboceptor 
of  the  goose  immune  serum  fails  in  our  experiment  to  bring  about  a 
deflection  of  complement  is  because  there  is  not  sufficient  affinity 
between  the  complements  of  pigeon  and  rabbit  serum  on  the  one 
hand  and  the  free  amboceptor  of  the  immune  serum  on  the  other. 


BACTERICIDAL  TEST-TUBE   EXPERIMENTS. 


135 


By  varying  the  experiment  I  have  succeeded  with  this  same  immune 
serum  in  producing  this  phenomenon  of  deflection  on  another  comple- 
ment, thus  furnishing  evidence  of  the  correctness  of  these  views. 
The  source  of  this  complement  was  an  active  normal  goat  serum 
which  in  itself  was  bactericidal  for  the  amount  of  organisms  em- 
ployed in  the  culture.  (See  Control  II.)  This  experiment  is  other- 
wise similar  to  the  preceding,  except  that  as  additional  control  tests 
the  corresponding  normal  sera  have  been  subjected  to  examination 
respecting  their  deflecting  power. 

TABLE  II. 
A. 


Amount  of  Culture. 

Amount  of  In- 
active   Goose 
Immune  Serum 
Directed 
against  Vibrio 
Metchnikoff. 
cc. 

Number  of  Colonies  on  a  Plate 
after  Completion  with 

0.3  cc.  Active 
Normal  Rabbit 
Serum. 

0.4  cc.  Active 
Normal  Pigeon 
Serum. 

j$v  cc.  of  a  one-day  bouillon  culture  ^ 

1.0 
0.3 
0.1 
0.03 
0.01 
0.003 
0.001 

0 
0 
0 
0 

10 

many  thous'd 

00 

0 
0 
0 
0 
0 
100 
00 

B. 


Amount  of  Culture. 

Amount  of 
Inactive    Goat 
Immune  Serum 
against  Vibrio 
Metchnikoff. 
cc. 

Number  of 
Colonies  on  a 
Plate  on  Com- 
pletion with 
0.3  cc.  Active 
Normal  Rabbit 
Serum. 

ttB  cc.  of  a  one-day  bouillon  culture  of  vibrio 

1.0 
0.3 
0.1 
0.03 
0  01 

00 
00 

0 
0 

o 

0.003 
0.001 
0.0003 

0 
0 

00 

Control     I.  E^S  cc.  bouillon  culture  +  2  cc.  0.85%  salt  solution  =  oo . 

"         II.  Xormal  active  rabbit-serum  0.3+j^  cc.  bouillon  culture =00. 

"       III.  0.4  cc.  active  pigeon-serum  ^-^  cc.  bouillon  culture  =00. 

"        IV.  Sterility  of  all  the  sera,  0. 


136 


COLLECTED  STUDIES  IN   IMMUNITY. 
TABLE  III. 


1 

2 

3 

4 

Amount  of 
the  Active 
Normal 

Amount 
of  the 
Inactive 

Number  of  Colonies  on  a  Plate  on  the  Addition 
of  the  Inactive  Sera  here  mentioned. 

Amount  of  Culture. 

Goat 
Serum, 
in  itself 
Bacteri- 
cidal. 

Immune 
and 
Normal 
Sera. 

Goat 
Immune 
Serum 
against 
Vibrio 

Normal 
Goat 
Serum. 

Goose 
Immune 
Serum 
against 
Vibrio 

Normal 
Goose 
Serum. 

cc. 

cc. 

Metchnikoff 

Metchnikoff 

yfoj  cc.  of  a  one- 
day  bouillon 
culture  of  vibrio 
Metchnikoff 

0.04 

1.0 
0.3 
0.1 
0.03 
0.01 

00 

sev'l  h'n'd 
0 
0 

sev'l  h'n'd 
0 
0 
0 
0 

00 
OC 
00 

sev'l  h'n'd 
0 

100 
0 
0 
0 
0 

Control      I.  7i^  cc.  bouillon  culture +  2  cc.  0.85%  salt  solution=oo. 

"         II.  Normal  active  goat-serum  0.04  cc. +5^  cc.  bouillon  culture  =  0. 
"       III.  Sterility  of  all  the  sera  =  0. 

The  principal  difference  in  this  as  compared  with  the  former 
experiment  is  that  the  goose  immune  serum  deflects  the  comple- 
ment even  more  strongly  than  does  the  goat  immune  serum. 

Thus  the  objection  that  the  deflection  of  complements  is  due  to 
agglutination  has  been  refuted  by  these  experiments  also.  The 
behavior  of  the  normal  sera  employed  as  controls,  whose  antibac- 
tericidal  power  even  in  amounts  of  1.0  cc.  is  very  slight,  will  be  spoken 
of  in  the  next  section. 

B.    Is  the  Deflection  of  Complements  due  to  Normal 
Anticomplements  ? 

The  deflection  of  complements  under  discussion  has  been  ascribed 
by  Metchnikoff 1  to  anticytases  normally  present.  This  objection 
falls  to  the  ground  if  it  can  be  shown  that  the  specific  immune  serum 
exhibits  a  constant  and  distinct  difference  in  comparison  to  other 
immune  sera  or  to  various  normal  sera.  It  is  entirely  immaterial 
if  the  normal  sera  also  show  this  phenomenon  to  a  slight  degree, 
e.g.  Table  III,  columns  2  and  4.  An  adequate  explanation  of  this 
has  already  been  furnished  by  Neisser  and  Wechsberg,  who  were  also 
the  first  to  describe  normal  anti complements.  Just  this  quanti- 
tative difference  between  immune  serum  and  normal  serum  is  one 


L'Immunit£  dans  les  Maladies  infectieuses,  page  313. 


BACTERICIDAL  TEST-TUBE  EXPERIMENTS.  137 

of  the  postulates  of  Ehrlich's  theory,  and  it  is  this  quantitative 
difference  which  constitutes  the  essential  point  in  the  deflection  of 
complements  described. 

The  following  table  includes  ten  different  goat  sera,  among  them 
three  bactericidal  immune  sera  (columns  2,  3,  4),  one  antitoxic  serum 
(column  5),  four  hsemolytic  immune  sera  (columns  6,  7,  8,  9),  and 
one  anticomplement  serum  directed  against  the  complements  of 
horse  serum  (column  10).  All  of  these  have  been  tested  as  to  their 
complement-deflecting  power  against  vibrio  Metchnikoff. 

The  experiment  has  been  slightly  modified  from  the  former, 
for  in  this  I  made  use  of  a  mixture  of  0.1  cc.  active  guinea-pig  serum 
(in  itself  not  bactericidal,  Control  II)  plus  0.01  cc.  inactive  goat 
immune  serum  (against  vibrio  Metchnikoff).  This  mixture  completely 
killed  the  amount  of  bacterial  culture  used,  namely,  1TrV(7  cc.  of  a 
one-day  bouillon  culture  of  vibrio  Metchnikoff  (see  Control  II).  De- 
creasing amounts  of  various  inactive  goat  sera  were  added  to  this, 
as  is  shown  in  columns  1  to  10. 

According  to  this,  the  Metchnikoff  immune  serum  exerts  a  specific 
action,  and  it  is  certainly  too  hazardous  to  assume  that  the  Metchni- 
koff goat  used  by  us  happened  to  possess  an  unusually  large  amount 
of  normal  anticomplement.  Furthermore,  it  is  possible,  as  I  shall 
show  later,  to  furnish  positive  proof  that  the  deflection  of  complement 
is  caused,  not  by  the  anti complements,  but  by  the  amboceptors; 
for  by  removing  the  amboceptors  it  is  possible  to  prevent  the  deflec- 
tion. The  following  experiment  furnishes  still  further  evidence 
against  Metchnikoff 's  view:  I  examined  the  serum  of  a  rabbit  before 
and  after  immunization  with  vibrio  Metchnikoff  and  found  that  the 
normal  serum  was  entirely  inactive,  whereas  after  eight  days  the 
immune  serum  of  this  animal  caused  strong  deflection.  In  these 
cases,  therefore,  it  will  not  do  to  ascribe  the  deflection  of  complements 
*to  a  normal  anticomplement.  That  normal  anticomplements  do 
occur  and  that  they  may  at  times  simulate  the  phenomenon  above 
described  is,  of  course,  possible  and  has  already  been  emphasized 
by  Neisser  and  Wechsberg.  In  such  cases  suitable  control  tests, 
above  all  the  absorption  method  described  in  the  next  section,  will 
guard  against  errors.  From  all  this  it  follows  that  the  phenomenon 
of  complement  deflection  which  can  be  observed  in  suitable  cases  is 
not  to  be  ascribed  to  the  presence  of  a  normal  constituent  but  to  one 
produced  by  immunization. 


138 


COLLECTED  STUDIES  IN   IMMUNITY. 


TABLE 


Amount  of 
Culture. 

Amount  of  the  Bacteri- 
cidal Serum  Mixture. 

Amount 
of  the 
Inacti- 
vated 
Goat 
Serum. 

cc. 

l 

2 

3 

Number  of  Colonies 

Normal 
Serum. 

Immune 
Serum 

against 
Vibrio 
Metchnikoff. 

Immune 
Serum 
against 
Vibrio 
Nord- 
hafen. 

TuVffCC-of  a  one- 
day  bouilion 
culture      o  f 
vibrio  Metch- 
nikoff 

0.1  cc.  active  normal 
guinea  -  pig     serum 
plus  0.01  cc.  inactive 
goat  immune  serum 
against  vibrio  Metch- 
nikoff 

1.0 
0.3 

\  0.1 
0.03 
I  0.01 

0 
0 
0 
0 
0 

almost  oo 

"          00 

00 

sev'l  hun'd 
0 

0 
0 
0 
0 
0 

Control    I.  YtfVu  cc.  bouillon  culture +  2  cc.  0.5%  salt  solution  =00. 

11       II.  Active  guinea-pig  serum  0.1  cc.  +ToW  cc.  bouillon  culture  =00. 


C.    Is  the  Deflection   of  Complements  Caused  by  Anticomplements 
Developed  by  Immunization? 

The  assumption  that  when  immunizing  with  bacteria,  antialexins 
develop  in  the  serum  of  the  animals  treated,  and  that  these  substances 
exert  an  antibactericidal  and  antihsemolytic  action,  is  made  by 
Gruber  l  solely  for  the  purpose  of  furnishing  an  explanation  for  the 
phenomenon  not  based  on  Ehrlich's  views.  Wechsberg2  has  very 
properly  pointed  out  that  Gruber's  assumption  completely  contradicts 
all  our  previous  experiences,  for  then  neither  by  active  nor  by  pas- 
sive immunization  should  we  benefit  the  organism  treated,  but  we 
should  even  injure  it.  The  evidence  on  which  Gruber  bases  this  new 
•conception  consists  in  hsemolytic  test-tube  experiments  in  which  he 
shows  that  bactericidal  immune  serum  hinders  the  haemolysis,  whereas 
the  corresponding  normal  serum'  does  not  do  so.  Wechsberg  in  a 
recent  study3  was  never  able  to  obtain  this  result,  even  with  the 
same  method  of  making  the  experiment  as  employed  by  Gruber. 
Similar  negative  results  were  obtained  by  H.  Sachs  of  this  institute, 
who  studied  a  number  of  immune  sera  for  this  purpose,  viz.,  immune 
sera  against  vibrio  Metchnikoff,  vibrio  Nordhafen,  staphylococcus, 

1  Wiener  klin.  Wochenschr.,  1901,  No.  50. 

2  Ibid. 

3  Ibid.,  1902,  No.  13. 


BACTERICIDAL  TEST-TUBE   EXPERIMENTS. 


139 


IV. 


4 

5 

6 

7 

8 

9 

10 

on  a  Plate  on  the  Addition  of  the  Inactive  Goat  Sera  here  mentioned. 


Serum 
against 
Staphylo- 
coccus 
Aureus. 

Serum 
against 
Staphylo- 
coccus 
Toxin. 

Serum 
against 
Rabbit 
Blood- 
cells. 

Serum 
against 
Sheep 
Blood- 
cells. 

Serum 
against 
Ox  Blood- 
cells. 

Serum 
against 
Human 
Blood-cells. 

Serum 
against 
Horse 
Serum. 

0 

sev'lhun'd 

0 

20-40 

many  thou. 

almost  oo 

100 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

Control  III.  Active  guinea-pig  serum  0.1  cc.  +0.01  cc.  inactive  goat  immune 
serum  against  vibrio  Metchnikoff  +nfor  cc-  bouillon  culture. 
IV.  Sterility  of  all  the  sera=0. 

erysipelas  of  swine,  hog  cholera,  dysentery.  I  am  unable  to  say 
what  the  cause  of  these  contradictory  results  may  be.  One  thing, 
however,  is  shown  thereby,  namely,  that  there  is  no  general  law  such 
as  Gruber  assumes,  and  that,  their  toxicity  taken  for  granted,  his 
experiments  constitute  rather  a  rare  exception  which  must  even  be 
regarded  as  an  unfortunate  coincidence. 

That  immunization  with  any  kind  of  bacteria  does  not  cause  the 
formation  of  anticomplements  with  a  general  antibactericidal  action, 
in  Gruber's  conception  is  seen  by  glancing  at  Table  IV,  columns  2, 
3,  and  4.  In  these  experiments  only  the  Metchnikoff  immune  serum 
exerted  a  complement-deflecting  action,  not,  however,  the  immune 
.sera  of  two  other  goats  immunized  with  vibrio  Nordhafen,  and  with 
staphylococcus  pyogenes  aureus. 

It  is  very  easy  to  prove  that  in  the  Metchnikoff  immune  serum 
the  active  factor  which  effects  this  anticomplementary  action  and 
which  develops  as  a  result  of  the  immunization  is  really  an  ambo- 
ceptor;  for  by  previously  adding  the  corresponding  dead  bacteria 
to  the  immune  serum  and  later  centrifuging,  the  amboceptor  of 
the  serum  is  abstracted.  It  can  then  be  shown  that  this  amboceptor- 
free  immune  serum  has  lost  all  its  power  to  deflect  complement, 
provided,  of  course,  a  sufficient  amount  of  bacteria  was  used.  It 
can  further  be  shown  in  this  wray  that  the  action  proceeds  quanti- 
tatively. Thus  if  decreasing  amounts  of  bacteria  are  employed 


140  COLLECTED  STUDIES  IN  IMMUNITY. 

to  absorb  the  amboceptors,  e.g.,  1,  J,  TV,  -fa  agar  culture,  it  will  be 
found,  for  instance,  that  the  whole  agar  culture  completely  abstracts 
the  amboceptor  from  the  serum;  one-quarter  of  the  culture  abstracts 
only  part  of  the  same,  smaller  amounts  still  less  corresponding  to 
the  amount  of  bacteria  added. 

But  by  this  method  I  was  able  to  bring  further  proof  of  the  specific 
action  of  the  immune  serum.  Thus  when  I  added  dead  Metchnikoff 
vibrios  to  the  Metchnikoff  immune  serum,  1  was  able  to  remove 
the  amboceptor;  the  serum  then  did  not  show  even  a  trace  of  deflect- 
ing action.  If,  however,  to  this  Metchnikoff  immune  serum  I 
added  other  bacteria  (vibrio  Nordhafen,  typhoid,  dysentery)  the 
serum  lost  none  of  its  power  to  deflect  complement,  because  the 
immune  body  of  Metchnikoff  immune  serum  is  anchored  only  by 
Metchnikoff  vibrios,  and  not  by  any  other  kind  of  bacteria. 

Such  an  experiment  is  reproduced  in  extenso  below.  For  certain 
reasons  to  be  explained  directly,  this  requires  a  tedious  and  complex 
method  of  procedure.  As  in  the  previous  experiment  I  employed  a 
mixture  of  active  guinea-pig  serum  and  inactive  Metchnikoff  immune 
serum  (from  a  goat),  which  sufficed  to  kill  the  amount  of  bacterial 
culture  employed  (Control  II).  To  this  mixture  are  added  decreasing 
amounts  of  the  native  inactive  Metchnikoff  immune  serum  of  a 
goat  (column  1).  The  same  immune  serum  previously  treated  with 
Metchnikoff  vibrios  is  shown  in  column  2;  treated  with  Nordhafen 
vibrios,  column  3;  with  typhoid  bacilli,  column  4;  and  with  dysentery 
bacilli,  column  5.  This  preliminary  treatment  with  bacteria  is  as 
follows:  Agar  cultures  of  the  various  bacteria  are  suspended  each 
in  2  cc.  0.85%  salt  solution  and  killed  by  heating  these  suspensions 
to  65°-70°  for  one  hour.  If  to  these  four  suspensions  we  were  now 
to  add  Metchnikoff  immune  serum  with  the  object  of  having  the 
immune  body  absorbed,  we  should  later,  on  centrifuging  to  remove 
the  bacteria,  encounter  great  difficulties,  it  being  impossible  in  this 
way  to  obtain  a  clear  fluid  free  from  bacteria.  The  Metchnikoff 
vibrios  alone  are  an  exception,  because  they  are  agglutinated  by  the 
corresponding  immune  serum.  Although,  according  to  Gruberr 
even  a  considerable  accumulation  of  bacteria  is  without  effect  on 
haemolysis,  in  my  bactericidal  experiments  I  met  with  the  annoying 
fact  that  such  large  amounts  of  bacteria  (the  centrifuged  fluid  is 
cloudy)  are  in  themselves  strongly  antibactericidal.  I  was  able 
to  overcome  this  difficulty  as  follows:  2.0  cc.  of  the  corresponding 
inactive  immune  serum  were  added  to  the  suspended  agar  cultures 


BACTERICIDAL  TEST-TUBE  EXPERIMENTS. 


141 


in  order  to  effect  agglutination;  thus  Metchnikoff  vibrios  to 
Metchnikoff  immune  serum,  Xordhafen  vibrios  to  Nordhafen 
immune  serum,  etc.  The  mixtures  were  kept  at  37°  C.  for  one  hour, 
diluted  to  25  cc.  with  salt  solution  (in  order  to  dilute  the  serum  as 
much  as  possible),  and  then  centrifuged.  The  fluids  were  poured 
off ;  the  sediments  consisting  of  agglutinated  bacteria  were  thoroughly 
shaken,  each  with  2J  cc.  inactive  Metchnikoff  immune  serum  and 
allowed  to  stand  for  1J  to  2  hours  at  37°  C.,  the  mixtures  being  occa- 
sionally shaken.  On  then  again  centrifuging  for  a  long  time,  I  was 
able  to  pour  off  a  clear  bacterial-free  fluid  which  was  used  for  the 
following  experiment  (columns  2  to  5). 

TABLE  V. 


| 

1 

2 

3 

4 

5 

JS 

Number  of  Colonies  on  a  Plate  on  the  Addi- 

5 

tion  of  Inactive  Goat  Serum  against 

91 

0 

Vbrio  Metchnikoff. 

Amount  of 
Culture. 

Amount  of  the 
Bactericidal  Serum 
Mixture. 

1.- 

1 

Previously  Treated  with  Dead 
and  Agglutinated 

£  2 

m 

ii 

| 

ti 
o  ^ 

is 

b.j 

1? 

1J 

II 

g 

& 

11 

1 

J~£ 

<5 

c 
i—  i 

X 

£ 

Q 

TGVTS   cc.    of    a 
one-day  bouil- 
lon culture  of 
vibrio  Metch- 

0.1 cc.  active  guinea- 
pig  serum  plus  0.01 
cc.     inactive    goat 
immune     serum 

fl.O 
1  0.5 

J0.25 

00 
00 
00 

0 
0 

0 

00 
00 
many 
thou'd 

00 
00 
many 
thou'd 

00 
00 

100 

nikoff 

against      vibrio 

0.1 

10-20 

0 

10-20 

10-20 

0 

Metchnikoff 

10.05 

0 

0 

0 

0 

0 

Control      I.  -n&nr  cc.  bouillon  culture +  2  cc.  0.85%  salt  solution  =  00. 

II.  0.1  cc.  active  guinea-pig  serum  +0.01  cc.  inactive  goat  immune 
serum  against  vibrio  Metchnikoff  +T^TS  cc-  bouillon  culture =0. 
"       III.  Sterility  of  all  the  sera  =  0. 

This  experiment  shows  that  by  previously  adding  dead  bacteria 
of  the  corresponding  species  and  then  using  the  centrifuged  serum, 
it  is  possible  to  remove  the  property  by  means  of  which  an  immune 
serum  when  in  excess  can  exert  a  complement-deflecting  action. 
This  absorption,  however,  did  not  succeed  with  three  other  species 
of  bacteria.  Hence  we  can  conclude  that  the  deflecting  agent  of 
the  immune  serum  is  a  substance  produced  by  immunization  and 


142  COLLECTED  STUDIES  IN  IMMUNITY. 

related  to  the  complement  on  the  one  hand  (complement  deflection !) 
and  to  the  corresponding  bacterium  on  the  other  (specific  absorption). 
It  is  therefore  an  amboceptor  in  Ehrlich's  sense,  and  not  an  anti- 
alexin  in  that  of  Gruber. 

The  results  of  my  experiments  may  be  summarized  as  follows: 

(1)  By  comparing  two  bactericidal  immune  sera  both  possessing 
a  strong  agglutinating  property,  while,  in  certain  combinations,  only 
one  manifested  the  phenomenon  of  deflection  of  complement,  the 
objection  was  controverted  that  this  deflection  is  due  to  the  mechanical 
action  of  agglutination. 

(2)  It  was  possible  to  show  in  several  different  ways    that  the 
deflection  of  complement  is  not  caused  by  a  constituent   of*  normal 
serum. 

(3)  It  was  directly  proven  that  the  deflecting  agent  of  the  immune 
serum    is    the    specific    amboceptor    (immune    body)    produced    by 
immunization. 

From  this  it  follows  that  the  amboceptor  merely  plays  the  role 
of  a  coupling  element  between  bacteria  and  complement  and  that 
the  property  of  "sensitizing"  (Bordet)  or  of  "preparing"  (Gruber) 
cannot  be  ascribed  to  it.  The  latter  assumptions  seem  to  be  irrecon- 
cilable with  the  phenomenon  of  deflection  of  complements  described 
by  Neisser  and  Wechsberg. 


XL    ACTIVE  IMMUNITY    AND   OVERNEUTRALIZED 
DIPHTHERIA    TOXINS.1 

By  Dr.  JULES  REHNS. 

INSTEAD  of  following  the  classical  method  of  immunizing  against 
diphtheria,  namely,  by  inoculating  the  toxin  in  gradually  increasing 
doses,  a  number  of  workers  have  attempted  to  produce  immunity 
by  inoculating,  either  from  the  outset  or  during  the  course  of  the 
immunizing  process,  mixtures  in  which  the  toxin  was  partly,  wholly, 
or  over  neutralized.  It  will  at  once  be  realized  that  these  methods, 
with  which  the  names  Babes,  Pavlovsky,  Arloing,  Madsen,  and  Kretz 
are  principally  associated,  possess  an  entirely  different  significance. 

Under  the  direction  of  Professor  Ehrlich  I  have  tried  to  see  whether 
active  immunity  could  be  conferred  upon  a  given  normal  organism  by 
the  injection  of  increasing  doses  of  diphtheria  toxin  mixed  with  one 
or  more  times  its  equivalent  of  antitoxin. 

Rabbits  weighing  about  2000  grams  were  used  and  these  were 
injected  with  mixtures  composed  of  a  toxin  L  and  the  Standard 
Serum  of  the  Institute. 

The  constants  of  this  poison,  determined  according  to  the  clas- 
sical methods  devised  by  Ehrlich  were  as  follows: 

(1)  The  amount  of  poison  which  just  corresponded  to  an  immu- 
nizing unit,  i.e.,  the  limit  of  no  action  whatever, 

L0=0.3  cc. 

(2)  The  amount  of  poison  which,  mixed  with  one  immunizing 
unit  of  serum,  was  just  sufficient  to  kill  the  animal,  the  so-called 
Lf  dose, 

Lt=0.45  cc. 

(3)  The  fatal  dose  for  a  rabbit  weighing  about  2000  grams,  deatk 
occurring  in  four  days: 

This  was  about  0.01. 

1  Reprinted  from  Compt.  rend,  de  la  Soc.  de  Biologie,  1901,  page  141. 

143 


144  COLLECTED  STUDIES   IN  IMMUNITY. 

Two  rabbits  were  inoculated  intravenously,  one  with 

3  units  serum  +  0.3  cc.  toxin, 

a  mixture  neutralized  about  three  fold  ;   the  other  with 
3  units  serum  +  0.45  cc.  toxin, 

a  mixture  about  doubly  neutralized. 

The  animals  received  daily  increasing  doses  from  the  5th  to  the 
18th  of  December,  1900,  at  the  end  of  which  time  the  total  amount 
of  toxin  they  had  received  was 

for  the  one     7.5    cc.  or  750  fatal  doses, 
for  the  other  4.27  cc.  or  429  fatal  doses. 

The  animals  showed  no  change  in  health  and  lost  no  weight. 

In  order  to  allow  the  excess  of  serum  introduced  time  to  be 
eliminated,  four  weeks  were  allowed  to  elapse  before  testing  the  serum 
for  its  antitoxic  strength. 

A  control  rabbit  treated  with  serum  alone  died  accidentally,  but, 
as  will  be  seen  from  the  results  of  the  experiment,  a  control  was 
superfluous. 

Both  rabbits  were  killed  Jan.  24,  1901,  and  1  cc.  of  the  serum 
was  mixed  with  one-quarter  a  Lt  dose.  The  test  animals  died  in 
twenty-four  hours.  By  decreasing  the  quantity  of  toxin  to  one- 
eighth  L-J-  dose,  death  occurred  in  forty  eight-hours. 

From  this  we  see  that  the  serum  of  these  animals  certainly  con- 
tains no  more  than  one-eighth  of  an  immunizing  unit,  an  amount 
which  at  once  eliminates  any  idea  of  a  passive  immunity. 

One  must  therefore  conclude  that  the  organism  of  a  normal 
rabbit  not  sensitized  through  previous  immunization  is  unable  to  break 
up  the  combination  of  diphtheria  toxin  with  antitoxin.  Not  a  trace  of 
this  toxin  is  free  at  any  moment,  and  the  strongest  doses  of  the  mix- 
ture are  destitute  of  any  injurious  effects.  Twenty  fatal  doses,  for 
instance,  were  given  at  the  beginning.  But  we  see  further  that  these 
mixtures  do  not  cause  even  the  slightest  production  of  antitoxin. 
We  must  therefore  conclude,  with  Arloing,  that  the  injection  of 
over-neutralized  toxin  is  absolutely  useless  for  purposes  of  immuni- 
zation. 

These  results  do  not  in  the  least  resemble  those  of  other  authors 
who  have  used  partially  neutralized  mixtures  in  which  toxons  and 
toxonoids  are  present  in  a  free  state.  So  far  as  immunizing  power 


DIPHTHERIA  TOXINS.  145 

is  concerned,  Madsen  has  found  that  these  substances,  though  abso- 
lutely free  from  pathogenic  action,  are  entirely  equal  to  pure  toxin. 
In  these,  then,  toxicity  and  immunizing  power  are  entirely  unasso- 
ciated.  These  facts  make  Ehrlich's  hypothesis  very  plausible,  ac- 
cording to  which  the  toxin  molecule  contains  separate  groups,  "  hapto- 
phore "  and  "  toxophore."  The  combination  of  the  former  with 
corresponding  groups  in  the  receptive  organs  furnishes  the  condi- 
tions necessary  and  sufficient  for  the  production  of  antitoxin  by 
these  organs. 

TRANSLATOR'S  NOTE. — Park  and  Atkinson  report  quite  different  results  in  a 
similar  set  of  experiments.  By  treating  horses  with  toxin  neutralized  threefold 
(for  guinea-pigs) ,  they  produced  a  considerable  amount  of  antitoxin.  Even  when 
the  toxin  was  neutralized  sixfold  there  was  a  slight  production  of  antitoxin. 
See  Proceedings  of  the  New  York  Pathological  Society,  1903. 


XII.    IS  IT  POSSIBLE  BY  INJECTING  AGGLUTINATED 

TYPHOID   BACILLI   TO   CAUSE  THE  PRODUCTION 

OF  AN  AGGLUTININ?1 

By  Prof.  M.  NEISSER,  Member  of  the  Institute,  and  Dr.  R.  LUBOWSKI,  formerly 
Assistant  in  the  Bacteriological  Division. 

ESPECIALLY  important  for  Ehrlich's  conception  of  the  chemical 
union  of  toxin  and  antitoxin  are  the  experiments  in  which  immu- 
nization of  animals  was  attempted  with  neutral,  and  therefore  non- 
poisonous,  toxin-antitoxin  mixtures.  Such  experiments,  inaugu- 
rated by  Babes,  were  recently  published  by  Kretz,2  among  others. 
At  first  it  appeared  to  this  author  that  he  could  really  immunize 
with  such  neutral  mixtures,  but  exact  reexamination  convinced  him 
of  the  contrary.  Jules  Rehns  3  also  was  unable  to  obtain  any  results 
with  neutralized  toxin-antitoxin  mixtures.  All  of  these  experi- 
ments showed  that  Ehrlich's  conception,  that  of  a  chemical  union 
of  toxin  and  antitoxin,  most  readily  sufficed  to  explain  the  facts. 

In  immunization  with  cellular  material  the  circumstances  are 
far  more  complex,  von  Dungern4  therefore  first  attempted  to 
rule  out  the  immunizing  action  of  the  injected  cells  (erythrocytes) 
by  simultaneously  injecting  the  Corresponding  immune  serum  obtained 
elsewhere.  This  mixture,  therefore,  was  neutral,  and  caused  no 
immunity  reaction.  Our  colleague,  Dr.  Sachs,  has  continued  these 
researches  at  the  suggestion  of  Professor  Shield,  and  will  report 
thereon  the  following  article. 

In  direct  contrast  to  v.  Dungern's  experiments  are  the  results 

1  Reprint  from  the  Centralblatt  f.  Bacteriologie  Parasitenkunde  und  Infec- 
tions Krankheiten,  Vol.  XXX,  1901,  No.  13. 

2R.  Kretz,  Ueber  die  Beziehungen  von  Toxin  und  Antitoxin,  Zeitschr.  f. 
Heilkunde,  1901,  No.  4. 

3  See  pages  143  et  seq. 

4  See  pages  36  et  seq. 

146 


AGGLUTINATED  TYPHOID  BACILLI.  147 

obtained  by  Jules  Rehns  l  by  the  injection  of  agglutinated  typhoid 
bacilli.  He  found  that  it  was  immaterial,  so  far  as  effect  was  con- 
cerned, whether  he  injected  the  typhoid  bacilli  agglutinated  or  not 
agglutinated.  An  entirely  similar  experiment  has  also  been  pub- 
lished by  Nicolle  and  Trenell.2 

Having  previously  and  independently  of  Rehns  busied  ourselves 
with  this  question,  and  having  seen  that  it  is  attended  with  con- 
siderable experimental  difficulties,  we  again  took  up  the  problem 
on  the  publication  of  Rehns'  article,  especially  because  of  the  theo- 
retical importance  of  the  subject.  Furthermore,  our  previous  experi- 
ences had  given  us  the  impression  that  Rehns'  results  were  not  gen- 
erally applicable. 

The  technique  of  our  experiments  was  as  follows:  The  typhoid 
culture  employed  was  an  old  laboratory  culture  especially  adapted 
to  agglutination  experiments.  Of  this,  one-day  agar  cultures,  sus- 
pended in  physiological  salt  solution  and  killed  by  exposure  for  one 
hour  to  60°- 70°  C.,  were  used  for  the  injections. 

The  preparation  of  the  agglutinated  typhoid  bacilli  was  most 
carefully  attended  to,  it  being  deemed  especially  important  to  fully 
satisfy  the  bacilli  with  the  agglutinin.  The  agglutinin  was  a  highly 
active  typhoid  agglutinin  derived  from  a  horse,  and  agglutinated 
even  in  dilutions  of  1:50,000;  only -in  the  last  experiments  was  a 
weaker  serum  used.  The  agglutinin  was  added  to  the  bacteria  in 
such  amounts  that  about  500-1000  times  the  amount  calculated 
to  be  necessary  was  used.  In  order  to  effect  as  firm  a  union  as  pos- 
sible between  bacilli  and  agglutinin  the  latter  was  allowed  to  act 
on  the  bacilli  for  one  hour  at  42°-44°  C.,  during  which  time  the  tubes 
were  shaken  every  ten  minutes  (at  times  with  glass  beads)  in  order 
to  loosen  the  larger  clumps  and  secure  the  penetration  of  the  agglutinin 
to  the  central  portions  of  the  clumps.  And  in  order  to  be  on  the 
safe  side,  we  centrifuged  the  bacteria  from  the  first  mixture  and 
repeated  the  saturating  process  in  the  same  manner.  After  the 
second  saturation  the  mixture  was  again  centrifuged,  filled  up  with 
salt  solution,  again  centrifuged,  and  then  washed  several  times. 
The  various  decantations  were  saved  and  tested  for  the  presence 
of  agglutinin;  the  last  washings  had  to  be  free  from  agglutinin. 

Concerning  the  amount  of  injected  bacilli  in  conformity  to  our 


1  J.  Rehns,  Compt.  rend,  de  la  Soc.  de  Biol.,  1000,  page  1058. 

2  Xicolle  et  Trenell,  Compt.  rend,  de  la  Soc.  de  Biol.,  1900,  page  1088. 


148 


COLLECTED  STUDIES   IN   IMMUNITY. 


previous  experience  we  took  two  agar  cultures  to  be  the  normal 
measure  for  one  rabbit.  Since  small  amounts  of  bacilli  were  lost 
through  the  centrifuging,  we  often  employed  somewhat  larger  amounts 
for  the  injection  of  the  agglutinated  bacilli;  while,  on  the  other 
hand,  the  control  animals  frequently  purposely  received  less  than 
two  agar  cultures.  This  was  done  to  meet  the  objection  that  the 
animals  injected  with  agglutinated  bacilli  had  received  fewer  bacilli 
than  the  control  animals.  But  just  in  these  control  animals  which 
therefore  received  different  amounts  it  was  seen  that  a  strict  paral- 
lelism between  the  amount  of  bacilli  injected  and  the  agglutinating 
value  produced  thereby  does  not  exist.  Many  animals  with  smaller 
doses  exhibited  higher  agglutinin  values  than  other  animals  with 
larger  doses,  as  is  seen  by  the  following  table  I. 

TABLE  I. 


Number  of  the 
Animal. 

Agglutinating 
Value  of  the 
Serum  Previous 

Amount  Injected. 

to  Injection. 

119 

0 

£  mass 

culture  subcutan. 

1:160 

118 

1:40 

1 

i  ( 

1:1280 

117 

1:40 

Tir 

i  ( 

1:320 

159 

0 

+  $  agar  culture 

1:1280 

160 

1:40 

tfr 

+t    ' 

1:1280 

162 

0 

x 

+  *    " 

1:2560 

The  injection  was  usually  subcutaneous,  a  few  times  intraperi- 
toneal.  The  blood  was  abstracted  from  the  ear  vein. 

Testing  the  agglutinating  value  of  the  serum  was  accomplished 
according  to  a  method  long  in  use  in  the  bacteriological  division,  as 
follows : 

The  serum  dilutions  (in  0.85%  salt  solution)  were  usually  1/2o, 
1Aor  Vso,  VIGO,  etc.;  finer  gradations  were  not  employed,  as  they 
are  of  no  value  in  measuring  the  agglutination.  The  culture  used 
was  a  living  20-hour  agar  culture  which  was  suspended  in  10  cc. 
of  bouillon.  To  each  serum  dilution,  whose  volume  was  1  cc.,  the 
same  amount  of  bacilli  was  added  (1  cc.  bouillon  culture),  so  that 
the  total  volume  of  each  specimen  was  2  cc.  Each  specimen  was 
then  poured  into  a  little  Petri  dish  and  placed  into  the  thermostat 
for  two  hours.  Thereupon  the  specimens  were  examined  with  the 
low  power  of  the  dry  objectives.  In  this  way  the  occurrence  of 
larger  or  smaller  clumps  is  very  distinctly  seen.  In  the  protocols 


AGGLUTINATED   TYPHOID   BACILLI. 


149 


only  "  complete  agglutination  "  and  the  "  positively  distinct  agglu- 
tination "  are  regarded  as  positive;  everything  at  all  doubtful  is 
regarded  as  not  agglutinated. 

The  first  question  was  on  which  day  following  the  injection  the 
maximum  agglutinating  value  was  to  be  expected  in  "the  serum  of 
the  animals.  Table  II  gives  a  resume  of  eight  animals  injected  with 
dead  typhoid  bacilli  and  examined  at  different  times. 

TABLE  II. 


Num- 
ber of 
the 

Aggluti- 
nating 
Value  of 
the 

Injected  Amount. 

Agglutinating  Value  on  the 

Ani- 

Serum 

1 

mal. 

Previous 

9th,  10th,1 

to  the 

5th  Day 

7th  Day 

or 

14th  Day 

15th  Day 

Injection 

llth  Day 

117 

1:40 

fa  mass  culture  subcut. 

1:80 

1:320 

118 

1:40 

i      " 

1:320 

1:1280 

119 

0 

i      "          " 

1:80 

1:160 

134 

1:160 

2  agar  cultures 

1:320 

1.640 

132 

0 

J    "         •• 

1:640 

1:640 

110 

? 

/T  mass  culture  + 
|  agar  culture    f 

1:640 

1:320 

1:160 

109 

TV  mass  culture  +       \ 
J  agar  culture    f 

1:2560 

1:1280 

1:320 

108 

TV  mass  culture  +       1 
£  agar  culture    / 

1:1280 

1:640 

1:640 

Four  of  these  animals  exhibited  a  lower  value  on  the  7th  (or  on 
the  5th)  day  than  on  the  14th  (or  on  the  10th)  day.  The  other  four 
animals  showed  a  decrease  or  no  change  at  all  in  their  agglutinating 
values  on  the  5th,  9th,  and  14th  (or  5th  and  10th)  days.  Hence 
if  on  the  7th  day  we  examined,  as  we  actually  did,  the  animals  which 
had  been  injected  only  with  dead  typhoid  bacilli,  we  were  not  sure 
that  we  should  strike  the  maximum  agglutinating  value.  That 
we  chose  this  time  nevertheless  is  explained  by  the  fact  that  this  simpli- 
fied the  investigations,  and  by  the  further  consideration  that  we 
did  not  need  the  highest  possible  values  in  these  control  animals. 

The  animals,  however,  in  which  we  were  compelled  to  strike  the 
maximum  value  are  seen  by  reference  to  Table  III  to  have  behaved 
differently.  Of  15  animals  which  had  been  injected  with  dead, 
agglutinated  typhoid  bacilli,  there  were  only  3  which  still  showed 
a  slight  increase  of  agglutinating  value  from  the  7th  to  the  14th 
(or  5th-9th)  day.  We  were  therefore  justified  in  withdrawing  the 


150 


COLLECTED   STUDIES  IN   IMMUNITY. 


blood  for  examination  of  all  the  animals  on  the  7th  day  after  the 
last  injection. 

TABLE  III. 


Value  on 

Number 

of  the 

Animal. 

5th  Day 

7th  Day 

9th  Day 

llth  Day 

13th  Day 

14th  Day 

15th  Day 

114 

1:80 

1:160 

115 

1 

1:80 

1:160 

111 

1:80 

1:166 

1:80 

103 

•Jj 

1:160 

1:160 

1:160 

104 

-Ha 

1:80 

1:80 

1:80 

106 

bc'"o 

1:160 

1:160 

1:160 

132 

c3_n 

0 

0 

181 

IB 

0 

0 

182 

••"a 

1:40 

1:40 

112 

0  43 

1:160 

1:160 

1:160 

116 

£  ^ 

0 

0 

131 

S3  "*•* 

0 

0 

133 

0 

0 

164 

1:20 

0 

105 

43 

H 

1:160 

1:80 

1:80 

It  may  further  be  mentioned  that  examinations  were  also  made 
on  the  29th  and  39th  day  ofter  the  injection,  in  which  however  a 
decrease  of  the  agglutinating  value  was  usually  found. 

Investigations  also  showed  that  injections  of  physiological  salt 
solution  in  bouillon  caused  no  variation  in  the  normal  agglutinating 
values. 

A  further  question  was  whether  and  to  what  degree  the  serum 
of  normal  untreated  rabbits  possesses  agglutinating  properties  oh 
typhoid  bacilli.  Out  of  17  rabbits  which  were  examined  for  this  pur- 
pose, 10  showed  no  agglutination  in  dilutions  of  1:20,  one  serum 
agglutinated  in  the  dilution  1 : 20,  but  no  higher,  5  others  in  1 : 40, 
but  no  higher,  and  only  one  agglutinated  even  in  a  dilution  of  1: 160. 
(See  Table  IV.) 

It  is  therefore  a  rare  exception  for  normal  rabbit  serum  to  still 
manifest  agglutinating  powers  on  typhoid  bacilli  in  a  higher  dilution 
than  1:40.  It  should  be  remarked  that  in  the  above  table  "0" 
has  always  then  been  put  down  when  the  agglutinating  value  of  the 
serum  in  a  dilution  of  1 :20=0;  for  the  examinations  began  with  this 
dilution. 


AGGLUTINATED  TYPHOID  BACILLI. 


151 


TABLE  IV. 
AGGLUTINATING  VALUES  OF  NORMAL  RABBIT  SERUM. 


Number  of  the 
Animal. 

Dilution  of  the  Serum. 

1:20. 

1:40. 

1:80. 

1:160. 

1:320. 

133 

0 

0 

0 

0 

0 

132 

0 

0 

0 

0 

0 

136 

0 

0 

0 

0 

0 

164 

0 

0 

0 

0 

0 

181 

0 

0 

0 

0 

0 

163 

0 

0 

0 

0 

0 

166 

0 

0 

0 

0 

0 

165 

0 

0 

0 

0 

0 

159 

0 

0 

0 

0 

0 

162 

0 

0 

0 

0 

0 

161 

+ 

0 

0 

0 

0 

114 

+ 

+ 

0 

0 

0 

160 

+ 

+ 

0 

0 

0 

182 

+ 

+ 

0 

0 

0 

117 

+ 

+ 

0 

0 

0 

118 

-f 

+ 

0 

0 

0 

134 

+ 

+ 

+ 

+ 

0 

We  now  come  to  the  experiments  proper.  In  the  first  of  these 
(Table  V)  a  series  of  rabbits  was  injected  with  agglutinated  typhoid 
bacilli,  while  a  control  series  was  injected  with  the  same  or  smaller 
amounts  of  non-agglutinated  bacilli.  This  comparison  shows  a  far 
higher  agglutinating  value  of  the  serum  of  the  control  animals  than 
that  of  the  other  animals. 

TABLE  V. 


Agglu- 

tinating 

Num- 
ber of 
the 

Value 
of  the 
Serum 

Injection  of 

Maximum 
Aggluti- 
nating 

Average. 

Animal. 

previ- 

Value. 

ous  to 

the  In- 

jection. 

Ill 

? 

1 

T^  mass  culture  +  1  agar  culture 

1:160 

112 

? 

ditto 

1:160 

103 

? 

•S  '§  '^ 

ditto 

1:160 

1:147 

104 

? 

•§•§.3 

ditto 

1:80 

105 

? 

"3;  >j-° 

ditto 

1:160 

106 

? 

<J 

•     ditto 

1:160 

< 

108 

? 

},  .A-oS- 

ditto 

1:1280 

} 

109 

? 

g  "§  2  J§  s| 

ditto 

1:2560 

1-1:1493 

110 

? 

*g3gj 

sV  mass  culture  +  i  agar  culture 

1:640 

J 

152 


COLLECTED   STUDIES  IN  IMMUNITY. 


The  next  question  was  whether  rabbits  really  react  at  all  to 
injections  of  agglutinated  typhoid  bacilli;  in  other  words,  whether 
the  normal  agglutinating  value  possibly  present  is  at  all  increased 
by  injections  of  agglutinated  typhoid  bacilli.  The  result  was  sur- 
prising, as  is  seen  in  Table  VI.  For  while  in  four  animals  no  increase 
occurred,  in  two  others  there  was  a  very  slight  increase,  and  in  four 
more  the  increase,  though  distinct,  was  insignificant  in  comparison 
with  that  in  six  animals  injected  with  non-agglutinated  typhoid. 

TABLE  VI. 


Agglu- 

Maximum 

tinating 

Agglu- 

Number 
of  the 

Value  of 
the  Serum 

Injection  of 

tinating 
Value 

Average. 

Animal. 

previous 

after 

to  the 

.the 

Injection. 

Injection. 

132 

0 

}  ^              2  agar  cultures  (intraperito- 

0 

neally) 

181 

0 

^              2  agar  cultures 

0 

116 

0 

>>             $  mass  culture 

0 

182 

1:40 

^  S              ditto 

:40 

164 

0 

•  JsS      i  mass  culture  +  %  agar  culture 

:20 

1  :  106 

163 

0 

g  JQ                             ditto 

:40 

166 

0 

ditto 

:320 

115 

0 

*jj         i  mass  culture 

:160 

161 

1:20 

|  mass  culture  +J  agar  culture 

:320 

114 

1:40 

<          i  mass  culture 

:160 

165 

0 

,          -g1^  mass  culture  +i  agar  culture 

:640 

] 

159 

0 

2-e      1     "  •      "      +*    " 

:1280 

162 
119 

0 
0 

18.2     ft    "        "     -B    " 

'   c3  ^        "  i         (  c             (  ( 

•*>£      * 

:2560 
:160 

!•  1  :  1093 

136 

0 

o  '•£       1  agar  culture 

:640 

160 

1:40 

^         -fa  mass  culture  +  f  agar  culture 

1:1280 

NOTE. — 1  mass  culture  equals  about  12  agar  cultures. 

With  this  the  main  portion  of  the  question  had  been  answered; 
for  these  experiments  already  showed  that  the  injection  of  agglutinated 
typhoid  bacilli  exerts  an  action  which  quantitatively  is  different 
from  that  following  the  injection  of  non-agglutinated  bacilli.  Never- 
theless even  the  agglutinated  bacilli,  although  their  injection  is 
often  wholly  without  effect,  in  many  cases  still  exert  a  stimulus  on 
the  formation  of  agglutinins  even  though  in  a  slight  degree.  This 
is  due  to  individual  peculiarities  of  the  animals  employed,  and  these 
we  have  not  thus  far  been  able  to  recognize  in  advance.  The  natural 
assumption  that  animals  which  already  normally  possess  agglutinins 
react  more  readily  to  the  injection  of  agglutinated  typhoid  bacilli 


AGGLUTINATED  TYPHOID  BACILLI.  153 

than  do  those,  which  do  not  normally  possess  agglutinins  has  not 
been  confirmed,  for  out  of  seven  animals  (Table  VI)  hi  whose  serum  no 
typhoid  agglutinin  could  be  demonstrated  previous  to  treatment, 
three  did  not  react  to  the  injection  of  agglutinated  typhoid  bacilli,  two 
reacted  feebly  and  two  very  distinctly.  On  the  other  hand,  out  of  three 
animals  in  which,  previous  to  treatment,  a  typhoid  agglutinin  could 
be  demonstrated,  two  reacted  distinctly  to  the  injection  of  agglutinated 
bacilli  and  one  not  at  all. 

Another  assumption  was,  that  in  the  animals  which  had  reacted 
but  feebly  or  not  at  all,  an  increase  of  the  sensitiveness  against  agglu- 
tinated bacilli  could  be  brought  about  artificially  by  repeated  injec- 
tions of  agglutinated  bacilli.  This  also  has  not  been  confirmed. 
Thus  three  animals  (Table, VII)  reacted  to  the  second  injection  of  agglu- 
tinated bacilli  just  as  little  as  they  did  to  the  first,  one  animal  reacted 
feebly,  as  it  had  done  previously,  and  only  two  animals  (Nos.  131  and 
133),  which  had  failed  to  react  to  the  first  injection,  reacted  distinctly 
to  the  second.  The  protocols  of  these  last  two  animals,  however,  point 
out  a  peculiarity.  On  the  first  occasion  these  animals  were  injected 
intraperitoneally  and  it  is  noted  that  at  this  time  the  intestine  was 
pricked.  The  first  injection  may  therefore  have  mostly  gone  into 
the  bowel  and  so  produced  no  effect.  The  second  injection  would 
then  have  really  been  the  only  effective  one.  These  two  cases  can- 
not therefore  be  used  to  prove  that  by  means  of  a  previous  injection 
of  agglutinated  bacilli  an  artificial  increase  of  the  sensitiveness  against 
a  subsequent  injection  of  agglutinated  bacilli  can  be  effected.  The 
previous  injection  of  agglutinated  bacilli,  however,  in  no  way  influences 
the  sensitiveness  against  non-agglutinated  bacilli,  as  is  shown  by 
the  four  control  animals  (Table  VII). 

Finally  experiments  were  made  regarding  still  another  assump- 
tion. It  was  conceivable  that  the  previous  injection  of  a  certain 
amount  of  non-agglutinated  bacilli  would  have  sufficed  to  bring  about 
a  sensitiveness  against  a  subsequent  inoculation  with  agglutinated 
bacilli.  This  assumption  also  has  not  been  borne  out.  Out  of  five 
animals  (Table  VIII)  which,  after  a  previous  injection  of  non-aggluti- 
nated typhoid,  received  an  injection  of  agglutinated  typhoid,  two 
showed  a  slight  increase  and  three  no  increase  in  agglutinating  value. 

It  follows  from  all  these  experiments  that  there  is  a  distinct  dif- 
ference between  the  injection  of  agglutinated  and  of  non-agglutinated 
typhoid  bacilli.  The  injection  of  non-agglutinated  typhoid  bacilli 
is  always  followed  by  an  increase  of  the  agglutinating  powrer.  This 


154 


COLLECTED  STUDIES  IX  IMMUNITY. 


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AGGLUTINATED  TYPHOID   BACILLI. 


155 


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156  COLLECTED  STUDIES  IN   IMMUNITY. 

increase  is  usually  very  great  and  only  rarely  slight.  The  injection 
of  agglutinated  typhoid  bacilli,  provided  that  attention  is  paid  to  a 
sufficient  saturation  with  agglutinin,  is  frequently  followed  by  no 
reaction,  often  by  a  slight  reaction,  and  rarely  by  a  marked  increase 
of  the  agglutinating  value.  This  reacting  power  depends  on  the 
individuality  of  the  animal  and  stands  in  no  relation  to  the  original 
agglutinating  value,  nor  can  it  be  influenced  artifically.  Furthermore, 
as  we  learned  from  a  special  experiment,  it  is  immaterial  whether 
the  immune  serum  used  to  agglutinate  the  typhoid  bacilli  is  derived 
from  the  same  or  from  another  animal  species. 

The  explanation  of  these  facts  is  not  difficult  provided  one  pro- 
ceeds on  Ehrlich's  theory.  According  to  this  the  agglutinin  consists 
of  thrust-off  cell-receptors.  As  a  result  of  their  seizure  by  the 
bacterial  receptors  they  have  been  produced  in  excess  and  give 
off  to  the  circulation.  They,  therefore,  possess  a  definite  relation 
to  the  corresponding  bacterial  receptors.  Hence  when  we  fully 
saturate  typhoid  bacilli  with  agglutinin,  we  cause  the  bacterial 
receptors  to  be  occupied,  and  are  then  as  little  able  to  cause  a  reaction 
with  these  bacteria  as  we  are  to  cut  with  a  sword  in  its  scabbard. 

If  then,  in  spite  of  this,  certain  animals  react  to  such  "occupied" 
typhoid  bacilli,  we  shall  have  to  assume  that  these  animals  possess 
the  power  to  dissolve  the  combination  of  agglutinin  and  bacterial 
receptor  and  thus  set  the  latter  free. 

This  action,  however,  never  proceeds  to  the  full  extent. 

Incomparably  more  important,  and,  as  it  appears  to  us,  explicable 
only  with  the  aid  of  Ehrlich's  chemical  views,  is  the  main  phenomenon, 
that  in  many  animals  no  reaction  whatever  follows  the  inoculation  of 
agglutinated  typhoid  bacilli;  that  therefore  in  many  cases  it  is 
possible  to  dispose  of  the  bacterial  group  giving  rise  to  the  agglutinin, 
by  causing  this  group  to  be  occupied  by  the  corresponding  agglutinin. 

SUBSEQUENT  NOTE. 

R.  Pfeiffer  and  Friedberger,1  through  recent  experiments  on  cholera  vibrios 
and  cholera  amboceptors,  have  obtained  results  which  are  in  gratifying  accord 
with  those  obtained  by  v.  Dungern,  M.  Neisser  and  Lubowski,  and  Sachs.2 
In  earlier  experiments  R.  Pfeiffer  3  had  found  that  the  bacterial  substance 
dissolved  in  the  peritoneum  through,  the  influence  of  the  cholera  immune  serum 

1  R.  Pfeiffer  u.  Friedberger,  Berl.  klin.  Wochenschr.,  1902,  No.  25. 

2  See  the  following  article. 

8  R.  Pfeiffer,  Deutsche  med.  Wochenschr.,  1901,  Nos.  50-51. 


AGGLUTINATED  TYPHOID  BACILLI.  157 

usually  still  excited  an  extraordinarily  strong  immunity  reaction,  a  phenomenon 
seemingly  in  contradiction  to  Ehrlich's  theory.  Further  experiments,  however, 
showed  that  when  very  high  doses  of  an  active  cholera  goat  serum  were  em- 
ployed, the  immunizing  action  was  almost  entirely  lost.  Of  especial  importance 
for  future  methodical  investigations  of  this  kind  is  the  fact  determined  by  these 
authors,  that  a  real  saturation  of  the  receptors  of  the  cholera  vibrios  requires 
a  surprisingly  high  multiple  of  the  amount  of  immune  serum  sufficient  to  dis- 
solve the  same  amount  of  cholera  vibrios.  7500  times  this  amount  does  not 
yet  satisfy  all  the  affinities  and  it  requires  enormous  doses,  up  to  3-4  million 
times,  to  completely  saturate  all  the  receptors. 


Xin.     IMMUNIZING    EXPERIMENTS    WITH    ERYTHRO- 
CYTES   LADEN   WITH  IMMUNE  BODY.1 

By  Dr.  HANS  SACHS,  Assistant  at  the  Institute. 

THE  interesting  experiments  of  v.  Dungern2  have  furnished 
further  proof  that  the  same  group  (receptor)  of  the  blood-cells  which 
in  haemolysis  combines  with  the  specific  immune  body  causes  the 
production  of  this  immune  body  within  the  organism,  v.  Dungern 
injected  rabbits  with  ox  blood  to  which  a  plentiful  amount  of  an 
immune  body  (obtained  from  rabbits  by  immunizing  with  ox  blood) 
had  been  added,  and  found,  as  was  to  be  expected,  on  the  basis  of 
the  side-chain  theory,  that  animals  so  treated  failed  to  produce 
any  immune  body  whatever. 

The  results  of  the  investigations  of  M.  Neisser  and  Lubowski3 
show  that  the  complete  inactivity  of  such  saturated  receptors — 
agglutinated  typhoid  bacilli — in  the  animal  body  is  not  at  all  a  general 
rule,  but  that,  on  the  contrary,  a  moderate  development  of  the 
immunity  reaction  occurs  even  with  such  mixtures  and  that  this 
depends  on  certain  individual  differences.  Hence  at  the  suggestion 
of  Prof.  Ehrlich  I  have  extended  the  experiments  of  v.  Dungern 
and  undertaken  blood-immunization  experiments  on  a  large  series 
of  animals.  The  results  obtained  lead  to  certain  modifications  of 
von  Dungern's  conclusions. 

The  method  of  these  experiments  must  be  guided  by  two  prin- 
ciples. To  begin,  it  is  important  that  the  receptors  of  the  injected 
blood  are  really  saturated,  for  even  a  very  slight  free  residue  might 
effect  an  immunity  reaction  in  the  animal  body.  And  yet  it  is  es- 
sential to  remove  any  possible  excess  of  immune  body,  because  this 

1  Reprint  from  the  Centralblatt  fur  Bacteriologie,  Parasitenkunde  und  In- 
fection Krankheiten,  Vol.  XXX,  1901,  No.  13. 

2v.  Dungern,  Muench.  med.  Wochenschr.,  1900,  No.  20.     See  also  page  56. 
3  See  the  preceding  article,  page  146. 

158 


IMMUNIZING  EXPERIMENTS  WITH  ERYTHROCYTES.        159* 

could  passively  reappear  in  the  serum  of  the  injected  animal  and  so 
simulate  an  active  new  formation  of  immune  body.  In  accordance 
with  this  the  experiments  were  made  as  follows :  Ox  blood  was  treated 
with  an  excess  of  inactive  serum  from  a  rabbit  which  had  been  im- 
munized with  ox  blood,  the  mixture  digested  at  37-40°  C.  for  half 
an  hour  and  then  centrifuged.  The  decanted  fluid  was  then  tested 
for  its  content  of  immune  body.  Only  when  this  test  proved  posi- 
tive, and  it  could  therefore  be  assumed  that  all  receptors  had  been 
saturated,  was  the  blood  so  treated  employed  for  injections.  But 
it  was  previously  repeatedly  washed  with  physiological  salt  solution 
in  order  to  remove  all  free  immune  body.  Finally  the  centrifuged 
sediment  was  made  up  to  its  original  volume.  The  course  of  such  an 
experiment  is  illustrated  in  the  following: 

100  cc.  ox  blood  are  mixed  with  25  cc.  inactive  immune  serum 
of  a  rabbit  which  has  been  immunized  with  ox  blood.  Of  this  im- 
mune serum,  0.0025  cc.  suffice,  when  complement  is  added,  to  just 
completely  dissolve  1  cc.  5%  ox  blood;  the  amount  employed,  there- 
fore, represents  five  times  the  amount  necessary  to  dissolve  the 
100  cc.  ox  blood.  After  the  mixture  has  remained  in  the  thermostat 
for  half  an  hour  it  is  filled  up  to  300  cc.  with  0.85%  salt  solution  and 
centrifuged.  The  first  decantation  is  tested  by  adding  it  in  decreas- 
ing quantities  to  1  cc.  5%  ox  blood  plus  0.4  cc.  normal  rabbit  serum 
(as  complement). 

The  following  results  are  obtained : 

1st.  Decantation:  1.5  cc.  complete  haemolysis. 

1.0  cc.  almost  complete  haemolysis. 
0.5  cc.       "  "  " 

0.25  strong  haemolysis. 
0.1    no 

From  this  it  must  be  concluded  that  the  blood-cell  receptors  are 
incapable  of  further  absorption ;  in  other  words,  that  they  have  been 
saturated. 

The  second  decantation  tested  in  this  same  manner  yields'  the 
following  result : 

2d.  Decantation:  3.0  cc.  strong. 

2.0  "  moderate. 
1.0  "  little. 
•    0.5  "  trace. 

It  therefore  contains  only  very  little  immune  body. 


160  COLLECTED  STUDIES  IN   IMMUNITY 

The  blood  is  once  more  washed  and  centrifuged  and  then  filled  up 
to  100  cc.  The  blood-cells  thus  saturated  with  immune  body  are  in- 
jected in  rabbits  intraperitoneally,  each  animal  receiving  25  cc.  of 
the  mixture. 

At  the  same  time  control  animals  are  injected  with  the  same 
amounts  of  normal  ox  blood. 

Usually  on  the  tenth  day  after  the  injection,  as  this  had  shown 
itself  the  most  favorable  time,  serum  was  withdrawn,  inactivated  and 
tested  for  its  content  of  immune  body  by  adding  it  in  decreasing 
quantities  to  1  cc.  5%  ox  blood  plus  sufficient  complement.  Either 
rabbit  serum,  0.4-0.5  cc.,  or  guinea-pig  serum,  0.1-0.15  cc.,  served 
as  complement,  for  these  are  equally  well  adapted  for  this  purpose. 
The  results  of  the  experiments  are  as  follows: 

Out  of  eight  rabbits  injected  intraperitoneally  with  ox  blood 
saturated  with  immune  body,  only  three  corresponded  to  the  requirements 
which  follow  from  von  Dungerris  results.  Their  serum,  tested  exactly 
like  the  immune  body,  failed  even  in  amounts  of  1.0  cc.  to  produce 
a  trace  of  haemolysis,  whereas  when  the  serum  of  the  corresponding 
control  animals  was  tested,  0.025  and  0.05  cc.  respectively  sufficed 
to  effect  complete  haemolysis. 

These  results  are  approached  very  closely  by  the  serum  of  a 
fourth  animal.  The  haemolytic  action  of  this  serum  compared  to 
that  of  the  serum  of  the  corresponding  control  animal  was  1:  <135, 
i.e.,  was  exceedingly  slight.  The  remaining  four  rabbits  had  pro- 
duced an  immune  body  in  greater  or  kss  amounts,  though  this  amount 
was  always  far  less  than  that  produced  by  the  corresponding  control 
animals.  When  the  absence  of  a  zone  of  marked  complete  solution 
rendered  it  impossible  to  make  an  exact  determination,  the  compari- 
son of  the  immune  body  values  of  the  sera  in  parallel  tests  was  accom- 
plished by  comparison  of  tubes  whose  colors  corresponded.  The 
amount  of  immune  body  possessed  by  these  animals  compared  to  that 
of  the  corresponding  control  animals  was  as  follows : 

(1)  1:5;  (2)  1:7;  (3)  1:10;  (4)  1:10. 

I  have  supplemented  these  experiments  with  a  smaller  series  of 
experiments  made  with  intravenous  injections.  In  these,  of  course, 
very  much  smaller  amounts  of  blood  were  used  for  injection  because 
when  the  blood-cells  loaded  with  immune  body  are  injected  directly  into 
the  circulation,  they  suffer  haemolysis  through  the  action  of  the  com- 


IMMUNIZING  EXPERIMENTS  WITH  ERYTHROCYTES.         161 

plement  present  in  the  serum,  causing  serious  symptoms  or,  with  larger 
amounts  of  blood,  fatal  results.  This  accords  with  the  phenomena 
observed  by  Rehns  l  when  he  injected  rabbits  which  had  been  immu- 
nized with  ox  blood,  intravenously  with  normal  ox  blood.  Only  two  of 
the  animals  1  employed,  namely  those  injected  with  7-8  cc.  blood,  re- 
mained alive  sufficiently  long.  In  one  of  these  only  traces  of  immune 
body  were  found  in  the  serum,  whereas  the  serum  of  the  other  animal 
effected  complete  solution  in  doses  of  0.05  cc.  In  the  serum  of  a 
control  animal  the  limit  of  complete  solution  was  0.01  cc.  These 
few  experiments  confirm  the  results  obtained  with  intraperitoneal 
injections,  that  blood-cells  saturated  with  immune  body  have  not  by  any 
means  always  lost  the  power  to  excite  a  certain  degree  of  immunity  reaction 
in  the  organism. 

Our  results,  therefore,  show  that  in  half  of  the  animals,  in  con- 
formity with  the  results  obtained  by  von  Dungern,  the  power  of  the 
blood  to  cause  an  immunity  reaction  is  lost,  owing  to  the  blocking  of 
that  particular  group  in  the  blood-cell  which  unites  with  the  immune 
body.  In  the  remaining  cases,  however,  the  specific  immune  body 
was  produced,  though  always  in  decidedly  less  amount,  since  only 
a  fifth  to  a  tenth  part  of  the  amount  appeared  that  was  produced  by 
the  control  animals.  This  apparently  unfavorable  portion  of  the  ex- 
periment shows  at  least  that  saturation  with  immune  body  exerts  a  marked 
restricting  influence.  These  results  agree  with  those  obtained  by 
Xeisser  and  Lubowski  with  injections  of  agglutinated  typhoid  bacilli. 

Furthermore,  like  Neisser  and  Lubowski,  in  an  animal  which  had 
not  reacted  to  the  injection  of  saturated  blood,  we  found  after  injec- 
tion of  the  same  amount  of  normal  blood  that  an  immune  body  of 
considerable  power  had  developed  in  the  serum.  The  complete 
solvent  dose  for  1  cc.  5%  ox  blood  amounted  to  0.005  cc.  serum  These 
last  experiments,  which  have  been  done  on  a  much  larger  scale  by 
Neisser  and  Lubowski  on  typhoid  bacilli,  indicate  that  the  failure  of 
antibodies  to  form  is  not  due  to  possible  individual  differences  in  the 
reacting  capacity  of  the  organism.  Considering  the  uniform  appear- 
ance of  immune  body  in  rabbits  treated  with  ox  blood  such  an  assump- 
tion would  have  lacked  all  probability. 

That  portion  of  the  experiments  in  which  the  injection  of  saturated 
blood-cells  was  borne  by  the  animals  without  producing  any  reaction, 
can  be  regarded,  as  has  been  done  by  von  Dungern,  as  a  complete  demon- 

1  Rehns,  Comp.  rend  de  la  Soc.  biol.,  1891,  No.  12. 


162  COLLECTED  STUDIES  IN   IMMUNITY. 

stration  that  the  groups  exciting  the  production  of  immunity  are  actually 
the  same  as  those  which  in  haemolysis  anchor  the  immune  body.  We 
have  seen,  however,  that  there  is  not  always  an  absence  of  reaction 
and  that  even  the  injection  of  the  same  saturated  blood,  which  in  one 
animal  fails  to  cause  a  production  of  immune  body,  is  followed  in 
another  animal  by  a  certain  low-grade  production  of  immune  body. 
The  cause  of  these  phenomena  can  only  be  that  certain  animals 
possess  the  individual  capacity  to  anchor  the  saturated  receptors  in 
spite  of  this  saturation.  We  do  not  know  the  mechanism  of  this 
action.  Two  factors  in  particular  come  into  consideration;  a  part 
of  the  immune  body  may  perhaps  be  destroyed  in  the  animal  body 
through  special  agencies  (oxidation?)  and  the  receptors  thereby  set 
free.  It  is  also  possible,  however,  without  assuming  a  destruction  of 
immune  body  to  explain  the  phenomenon  in  the  sense  of  Ehrlich's 
views,  by  assuming  a  higher  affinity  of  the  tissue  receptors  present  in 
the  animal  body,  which  receptors  then  would  be  able  to  break  up  the 
union  of  blood-cell  receptors  and  immune  body,  and  draw  the  blood- 
cell  receptors  unto  themselves.1 

Whichever  of  these  explanations  is  the  correct  one,  our  experiments 
certainly  show  one  thing,  that  the  dissolution  of  the  blood-cell  receptor 
combination  is  never  a  complete  one.  Merely  a  portion  of  the  groups 
is  concerned,  for  only  by  this  partial  dissolution  is  the  fact  (determined 
by  Neisser  and  Lubowski,  as  well  as  by  us),  to  be  explained  that  a 
very  slight  degree  of  immunity  reaction  is  produced  by  the  injection  of 
saturated  receptors. 

Hence  even  in  the  cases  running  an  apparently  unfavorable 
course,  only  a  part  always  of  the  receptors  exert  their  action.  This 
portion  of  the  experiments  may  therefore  also  be  used  as  a  sup- 
port for  the  side-chain  theory. 

1  A  similar  assumption  must  be  made  in  order  to  explain  certain  forms  of 
over-sensitiveness  studied  particularly  by  v.  Behring,  in  which,  despite  a  large 
excess  of  antitoxin,  very  small  doses  of  toxin  cause  death.  The  most  ready 
explanation  is  that  here,  in  contrast  to  the  behavior  in  normal  animals,  the 
toxinophile  receptors  possess  a  pathologically  increased  avidity  by  which  they 
are  enabled  to  break  up  the  neutral  toxin-antitoxin  mixture  (which  cannot  be 
broken  up  by  normal  cells)  and  take  up  the  toxin  thus  set  tree. 


XIV.  CONCERNING  THE  ESCAPE  OF  HEMOGLOBIN 
FROM  BLOOD  CELLS  HARDENED  WITH  CORROSIVE 
SUBLIMATE.1 

By  HANS  SACHS,  Assistant  at  the  Institute. 

THE  following  study  was  undertaken  on  reading  the  results  of 
investigations  carried  on  by  Matthes2  on  the  role  of  the  immune 
body  (amboceptor)  in  haemolysis.  The  peculiarity  of  his  very  inter- 
esting results  demands  a  thorough  study  of  the  factors  concerned. 

The  facts  there  brought  out  have  been  confirmed  by  us,  but  the 
results  of  our  study  have  led  us  to  regard  these  facts  in  an  entirely 
different  light.  As  a  result  of  numerous  earlier  experiences  with 
pepsin,  pancreatin  and  papain  we  can  confirm  the  observation  that 
normal  as  well  as  sensitized  red  blood-cells  (i.e.  cells  loaded  with 
immune  body)  cannot  be  attacked  by  digestive  ferments.3  With 
digestive  experiments  with  pepsin  and  pancreatin,  to  be  sure,  the 
difficulty  exists  that  the  amounts  of  HC1  and  alkali  respectively  which 
represents  the  optimum  of  action,  are  in  themselves  not  indifferent 
for  the  blood-cells.  With  these  ferments  one  is  therefore  forced  to 
work  under  relatively  unfavorable  conditions. 

Matthes  killed  the  blood-cells  by  means  of  Hay  em's  solution 
(which,  as  is  well-known,  contains  J%  mercuric  chloride)  and 
found  that  blood-cells  so  treated  were  readily  dissolved  by  means  of  active 
pancreas  fluid.  These  fixed  blood-cells,  which  are  no  longer  sus- 
ceptible to  the  destructive  action  even  of  distilled  water,  are  dissolved 
by  the  specific  haemolytic  serum  and  even  by  their  own  normal  serum. 

1  Reprint  from  the  Muenchener  med   Wochenschr  ,  1902,  No.  5. 

2  M.  Matthes,   Experimenteller  Beitrag  zur  Frage    der   Hamolyse,  Muench. 
med.  Wochenschr.,  1902,  No    1. 

' 3  According  to  recent  investigations  of  Dr,  Morgenroth,  the  interesting  intes- 
tinal ferment,  erepsin,  described  by  Cohnheim  and  by  him  kindly  placed  at  our 
disposal,  is  also  not  able  to  attack  sensitized  blood-cells. 

163 


164  COLLECTED  STUDIES  IN  IMMUNITY. 

Although  we  can  entirely  confirm  these  statements,  we  cannot  accept 
Matthes'  view,  according  to  which  the  solution  of  the  fixed  blood- 
cells  by  pancreatin  is  conceived  as  a  digestion,  the  Hay  em  solution 
acting  somewhat  like  an  immune  body.  The  striking  fact  that 
the  fixed  blood-cells  dissolve  even  in  their  own  serum  appeared 
to  us  rather  to  be  the  result  of  the  union  of  the  mercuric  chloride 
(which  adhered  to  the  blood-cells  and  prevented  this  solution)  with 
the  albumin  of  the  serum.  The  experiments  made  in  this  direc- 
tion at  the  suggestion  of  Prof.  Ehrlich  have  completely  confirmed 
this  view. 

Following  the  procedure  of  Matthes,  I  employed  rabbit  blood 
which,  freed  from  serum,  was  mixed  with  Hayem's  solution  in  the 
proportion  of  1:4.  After  standing  a  short  time,  the  blood  was  cen- 
trifuged  and  then  washed  three  or  four  times  with  0.85%  salt  solu- 
tion. Finally  a  5%  suspenson  of  the  fixed  blood-cells  in  .85% 
salt  solution  was  prepared.  The  corresponding  control  was  made 
with  normal  5%  rabbit  blood. 

In  the  experiments  1  cc.  of  the  5%  blood  mixtures  was  used;  the 
fluid,  after  the  addition  of  the  reagent  being  made  up  to  2  cc.  with 
physiological  salt  solution.  It  was  found  that  not  only  fresh  rabbit 
serum,  but  even  rabbit  serum  which  had  been  inactivated  by  half  an 
hour's  heating  to  56°  C.,  as  well  as  rabbit  serum  which  had  been  diluted 
with  ten  volumes  of  physiological  salt  solution  and  then  boiled  one  hour, 
was  still  able  to  cause  solution  of  the  fixed  rabbit  blood-cells;  0.075 
cc.  serum  causing  complete  and  almost  instantaneous  solution.  In 
this  case  the  toxic  action  of  the  serum  can  hardly  be  thought  of. 
The  experiment  indicated  rather  that  other  kinds  of  influences  are 
the  cause  of  this  curious  phenomenon.  If  the  conception  is  correct 
that  we  are  dealing  with  a  combination  of  the  mercury  with  the 
serum,  it  should  be  possible  also,  with  other  means  which  abstract 
the  mercury,  to  cause  a  solution  of  blood-cells  fixed  with  Hayem's 
solution.  As  a  matter  of  fact  this  can  very  easily  be  done.  I  chose 
potassium  iodide  and  sodium  hyposulphite  for  this  test  and  found 
that  extremely  small  amounts  of  these  substances  cause  immediate 
solution  of  the  fixed  blood.  0.00075  cc.  of  a  20%  KI  solution  in 
physiological  salt  solution  or  0.00025  cc.  of  a  similar  hyposulphite 
solution  sufficed  to  completely  dissolve  1  cc.  of  our  5%  fixed  blood 
suspension.1  This  positively  shows  that  the  function  of  the  serum 

1  With  normal  rabbit  blood,  1000-2000  times  the  amount  of  KI  or  of  hypo- 
sulphite solution  still  acts  indifferently. 


ESCAPE  OF   HEMOGLOBIN   FROM   BLOOD  CELLS.  165 

albumin  in  the  experiments  made  by  Matthes  is  that  which  we  assumed. 
It  must  therefore  be  concluded  that  the  blood-cells  treated  with 
Hayem's  solution  do  not  dissolve  in  water  because  the  mercuric 
chloride  with  which  they  have  combined  prevents  the  escape  of  the 
haemoglobin.  The  cause  of  this  may  be  that  the  soluble  substances, 
e.g.  the  haemoglobin,  form  an  insoluble  combination  with  the  mer- 
curic chloride;  it  is  sufficient,  however,  to  assume  that  the  limiting 
membrane  of  the  discoplasma  becomes  denser  through  the  deposited 
mercury  salt  and  so  prevents  the  diffusion  of  the  blood  coloring- 
matter.  Be  this  as  it  may,  certainly  all  agencies  which  break  up  the 
mercury  combination  will  cause  an  immediate  solution  of  the  hae- 
moglobin. The  reason  for  this  is  that  the  discoplasma,  which  in  the 
living  state  hinders  the  diffusion  of  haemoglobin,  has  been  killed 
by  the  sublimate  treatment, 

From  this  it  is  easily  seen  that  the  solution  of  the  fixed  blood 
by  means  of  pancreatin  as  it  is  described  by  Matthes,  is  not  to  be 
regarded  as  a  species  of  digestion.  Every  such  ferment  solution 
contains  enough  albumin  to  explain  the  action  according  to  our 
view.  I  was  able  to  confirm  this  by  the  experiment  in  which  the 
haemolytic  action  (observed  by  us  also)  of  neutral  pepsin  and  pan- 
creatin solutions  was  exerted  in  like  manner  when  the  solution  had 
previously  been  heated  to  95°  C.  for  1  hour. 

In  conclusion  it  may  be  remarked  that,  after  fixation  with  J% 
mercuric  chloride  solution  in  physiological  salt  solution  instead  of 
with  Hayem's  fluid,  the  blood-cells  behaved  in  exactly  similar  fashion, 
as  was  a  priori  to  be  expected.  The  control  tests  made  at  the  same 
time  with  normal  blood  gave  negative  results  in  all  the  experiments. 
On  the  other  hand  with  solanin,  a  substance  which  dissolves  normal 
blood  even  in  enormous  dilutions,  haemolysis  of  fixed  blood-cells 
could  not  be  effected  even  though  large  doses  were  employed.  In  this 
substance  the  necessary  albumin  is  wanting  and  the  dead  blood- 
cells  are  no  longer  vulnerable  to  the  action  of  the  blood  poison. 

To  sum  up,  we  may  say  that  in  the  blood-cells  hardened  with 
Hayem's  solution  it  is  merely  the  chemically  bound  r.iercuric  chloride 
which  hinders  the  escape  of  the  haemoglobin.  All  agents  which  are 
capable  of  attracting  this  salt  to  themselves,  i.e.  to  "  de-harden  "  the 
blood-cells,  cause  the  immediate  escape  of  haemoglobin. 

Hence,  although  the  observations  of  Matthes  are  extremely  inter- 
esting in  themselves,  they  possess  no  value  for  the  doctrine  of  hae- 
molysis. On  the  other  hand  it  would  seem  as  though  they  might 


166  COLLECTED  STUDIES  IN  IMMUNITY. 

be    applied    to    a    method    of    detecting    smallest    amounts    of 
mercury. 

SUBSEQUENT  ADDITION. — In  a  recent  communication  (Muench.  med.  Wo- 
chenschr.  1902,  No.  17)  Matthes  has  completely  confirmed  the  results  of  our 
experiments  so  far  as  mammalian  blood-cells  are  concerned.  The  fact  that 
other  species  of  blood,  such  as  frog  blood  studied  by  Matthes,  after  hardening 
with  mercuric  chloride,  do  not  give  up  their  haemoglobin  even  in  fluids  rich  in 
albumin  does  not  affect  our  view,  but  only  points  to  a  high  degree  of  hardening 
of  the  frog-blood  stromata  which  does  not  permit  the  escape  of  the  haemoglobin 
even  in  the  presence  of  substances  abstracting  mercury.  We  did  not  deny 
that  the  stromata  could  be  digested  by  means  of  proteolytic  ferment.  Our 
objection  was  directed  only  to  regarding  the  escape  of  haemoglobin,  an  indi- 
cation of  a  digestion,  or  of  digesting  complements. 


XV.  A  CONTRIBUTION  TO  THE  STUDY  OF  THE  POISON 
OF  THE  COMMON   GARDEN  SPIDER.1 

By  Dr.  HANS  SACHS,  Assistant  at  the  Institute. 

THE  studies  in  haemolysis,  constantly  keeping  pace  with  the  develop- 
ment of  the  doctrine  of  immunity,  have  shown  that  besides  the  usual 
blood  poisons  sharply  denned  chemically,  there  is  another  group 
of  haemolysins  of  animal  or  vegetable  origin  which  exert  their  damag- 
ing influence  like  the  toxins,  by  combining  with  certain  definite 
groups  of  the  protoplasm.  Included  in  this  are  snake  venom,  numer- 
ous bacterial  secretions  such  as  tetanolysin  and  staphylolysin,  tox- 
albumins  of  higher  plants,  such  as  crotin.  Besides  this  there  is 
the  endless  series  of  haemolysins,  both  normal  and  those  produced 
at  will  by  immunization,  which  are  found  in  the  blood  serum. 

Of  the  highest  importance  for  the  conception  of  the  similarity 
of  these  blood  poisons  was  the  fact  that  only  such  blood-cells  are  sen- 
sitive to  these  hcemolysins  which  are  capable  of  anchoring  them.  This 
fundamental  law,  which  was  first  recognized  and  clearly  formulated 
by  Ehriich  and  Morgenroth2  has  constantly  been  confirmed,  espe- 
cially in  the  study  of  the  serum  hsemolysins  artificially  produced. 
As  a  result  of  this  the  mode  of  action  of  these  poisons  as  well  as  of  the 
toxins  has  been  conceived  from  the  standpoint  of  the  side-chain  theory. 
"  *  *  *  the  prerequisite  and  the  cause  of  the  poisonous  action  in 
all  these  cases  is  the  presence  in  the  blood-cells  of  appropriate  receptors 
(side  chains)  which  fit  into  the  haptophore  groups  of  the  toxin;  con- 
versely, therefore,  there  is  an  intimate  connection  between  natural 
immunity  and  the  absence  of  receptors."  (Ehriich.) 

It  is  evident  that  the  study  of  the  combining  relations  of  the 
toxin-like  blood  poisons  is  of  great  significance  for  the  study  of  the 

1  Reprint  from  Beitrage  zur  chemischen  Physiologic  u.  Pathologic,  Vol.  II, 
No.  1-3. 

J  See  page  1  et  seq.  . 

167 


168  COLLECTED  STUDIES  IN  IMMUNITY. 

causes  of  this  poisonous  action.  Such  a  study,  moreover,  is  calcu- 
lated to  extend  our  knowledge  of  the  receptors  and  their  physio- 
logical distribution  in  the  animal  kingdom.  While  examining  an 
extract  derived  from  the  common  garden  spider  (Epeira  diadema) 
I  found  in  it  a  hsemolysin  which  showed  itself  particularly  well 
adapted  to  researches  in  this  direction. 

The  description  of  a  complete  experiment  will  give  an  idea  of 
the  method  of  obtaining  and  testing  this  poison. 

A  garden  spider  weighing  1.4  grams  is  rubbed  up  with  5  cc.  toluol  water 
containing  10%  NaCl  and  the  fluid  kept  in  the  refrigerator  for  twenty-four 
hours.  Then  water  is  added  to  make  the  total  volume  25  cc.  and  the  mixture 
filtered  (or  centrif uged) .  The  haemolytic  experiments  are  made  in  the  usual 
manner  with  this  cloudy,  brownish-yellow  nitrate.  Decreasing  amounts  of 
the  poison  solution  are  placed  in  a  series  of  test-tubes,  each  of  which  is  then 
filled  up  to  1.0  cc.  with  physiological  (0.85%)  salt  solution.  Each  tube  now 
receives  one  drop  of  undiluted  blood  or  1  cc.  of  a  5%  suspension  of  blood  in 
physiological  salt  solution.  The  specimens  are  kept  in  the  incubator  at  37°  C. 
for  two  hours,  and  then  in  the  refrigerator  until  the  following  day  when  the 
amount  of  solution  is  determined.  The  blood  employed  was  always  centrifuged 
and  washed  in  order  to  remove  the  adherent  serum  and  so  exclude  any  possible 
disturbance  from  that  source. 

The  Arachnolysin,  as  we  may  designate  the  active  principle  of 
the  poison  solution,  causes  solution  of  the  sensitive  blood-cells  even 
at  room  temperature;  when  present  in  certain  proportions,  solu- 
tion occurs  almost  instantaneously.  In  this  respect,  arachnolysin  is 
somewhat  analogous  to  snake  venom,  while  it  differs  therein  from 
the  haemolysins  of  blood  serum,  hi  which,  as  is  well  known,  actual 
haemolysis  is  preceded  by  a  longer  or  shorter  period  of  incubation. 
The  more  exact  determinations  on  different  species  of  blood  were 
made  in  the  usual  manner  and  yielded  the  results  shown  in  the  fol- 
lowing table.  The  amounts  of  arachnolysin  given  in  the  table  refer 
to  the  original  solution,  containing  28%  of  spider  substance. 

As  can  be  seen  from  the  table  we  are  here  dealing  with  a  hcemolysin 
of  extraordinary  power,  the  action  of  which  on  the  individual  species 
of  blood,  however,  is  very  variable.  Thus  a  number  of  species  of  blood 
are  destroyed  even  in  dilution  of  1 : 1000  or  1 : 10000  (this  refers  to 
the  original  poison  solution);  others  remain  unaffected  even  by  large 
amounts  of  poison.  Next  to  rat  blood,  the  most  sensitive  was 
rabbit  blood,  for  0.0001  cc.  of  the  original  solution,  i.e.,  0.000028  g. 
spider  substance,  sufficed  to  completely  dissolve  0.05  cc.  blood 
(=200,000,000  blood-cells).  A  garden  spider  weighing  1.4  g.  there- 


THE   POISON   OF  THE  COMMON   GARDEN  SPIDER. 


169 


Arachnolysin. 

Haemolytic  Action  on  the  Blood  of 

Rabbit. 

Rat. 

Mouse. 

Man. 

cc. 

1/1000 

1.0 

OTC 

complete 

complete 

complete 

complete 

.75 

0.5 

(  i 

<  < 

it 

0.35 

1  1 

t  f 

almost  complete 

0.25 

if 

almost  complete 

do. 

0.15 

(t 

do. 

moderate 

1/10000 

1.0 

(  t 

i  i 

i  ( 

0.75 

almost  complete 

almost  complete 

strong 

little 

0.5 

strong 

strong 

1  1 

trace 

Hsemolytic  Action  on  the  Blood  of 

A            U         1 

_s. 

A  r&c  tm  o  1  y  o  j.  u.. 

Ox. 

Goose. 

Gp£:a~  Horse- 

Sheep.      Dog. 

cc. 

1/1000 

1.0 

f\     JK 

complete 

strong 

0000 

u  .  <  o 
0.5 

almost  complete 
strong 

0.35 

little 

No   haemolysis   even   with 

0.25 

trace 

larger  amounts 

0.15 

0 

1/10000 

1.0 

— 

0.75 

— 

moderate 

0.5 

—  — 

1  1 

fore  contains  sufficient  poison  to  completely  destroy  2.5  liters  rabbit 
blood.  Remembering  that  only  an  extremely  small  part  of  the 
spider's  weight  is  made  up  by  the  active  poisonous  constituent, 
and  even  assuming  that  the  content  of  arachnolysin  amounts  to  1%, 
we  see  that  this  enormous  activity  indicates  that  the  arachnolysin  belongs 
to  the  class  of  blood  poisons  which  exert  a  powerful  action  after  the  man- 
ner of  the  toxins. 

The  same  is  indicated  by  the  marked  instabilty  of  the  active 
principle.  Heat  readily  destroys  the  arachnolysin,  although  a  higher 
degree  is  necessary  than  for  other  haemolysins.  Heating  to  56°  C. 
for  40  minutes  does  not  affect  the  poison  solution,  and  at  60°  C. 
only  a  very  slight  reduction  of  action  is  noticed.  Complete  destruc- 
tion does  not  occur  until  the  poison  is  heated  to  70°-72°  C.  for  40 
minutes.  Arachnolysin  is  easily  preserved  by  the  addition  of  glyc- 
erine, showing  no  reduction  in  activity  even  after  months. 

Experiments,  designed  to  show  whether  normal  sera  possess  an 
inhibiting  action  on  haemolysis  due  to  spider  poison,  have  had  nega- 


170  COLLECTED  STUDIES  IN  IMMUNITY. 

tive  results;  the  sera  of  man,  rabbit,  horse,  pig,  dog,  rat,  guinea- 
pig,  goat,  sheep,  ox,  goose,  and  pigeon,  inactivated  by  heating  to 
56°  C.  in  order  to  eliminate  any  possible  solvent  action,  were  unable 
even  in  amounts  of  1.0  cc.  to  protect  rabbit  blood  against  just  a  com- 
plete solvent  dose  of  arachnolysin. 

On  the  other  hand,  the  study  of  the  poison's  behavior  toward 
sensitive  and  insensitive  cells  has  yielded  results  of  special  interest 
in  connection  with  the  receptor  theory.  Certain  species  of  blood, 
such  as  dog  or  guinea-pig  blood,  have  shown  themselves  immune 
to  the  spider  poison.  This  presents  the  most  favorable  conditions 
for  studying  the  relations  between  the  binding  of  poisons  and  their 
action.  This  point,  as  we  have  seen,  is  of  the  greatest  importance 
for  the  view  that  serum  haemolysins  are  toxin-like  bodies. 

If  arachnolysin  is  a  blood-poison  whose  action  is  due  to  the  anchor- 
ing of  a  certain  haptophone  group  to  a  receptor  of  the  sensitive  blood- 
cell,  and  if,  corresponding  to  this,  the  immunity  of  certain  species 
of  blood  is  due  to  a  lack  of  appropriate  receptors,  it  follows  that 
the  sensitive  blood-cells  must  be  able  to  bind  the  active  principle 
of  such  a  poison  solution,  while  the  insensitive  cells  leave  it  entirely 
unaffected. 

So  far  as  the  insensitive  bloods  are  concerned,  the  method  of 
making  the  experiment  is  very  simple.  Dog  blood  is  mixed  with  a 
certain  quantity  of  arachnolysin,  kept  in  the  incubator  for  an  hour 
and  frequently  shaken.  Thereupon  the  blood,  which,  of  course,  is 
unchanged,  is  separated  by  means  of  a  centrifuge.  The  decanted 
fluid,  compared  with  the  original  material,  shows  not  the  least  diminu- 
tion of  its  solvent  power  on  rabbit  blood-cells.  This  shows  that  the 
insensitive  dog  blood  is  not  able  to  bind  the  arachnolysin. 

In  the  case  of  the  sensitive  blood-cells,  the  demonstration  of  the 
combining  power  is  much  more  difficult,  for  these,  when  tested  in 
a  similar  manner  are  dissolved,  so  that  it  is  impossible  to  separate 
blood-cells  and  fluid.  We  can  then  only  operate  with  the  laky  blood 
solution,  the  inactivity  of  which  permits  of  no  direct  conclusion 
that  a  binding  of  the  poison  by  means  of  receptors  had  occurred. 
Furthermore,  if  the  poison  solution  has  lost  its  power  as  a  result 
of  the  action  already  exerted,  there  is  no  means  by  which  this  can 
be  determined.  It  was  necessary,  therefore,  to  employ  blood-cell 
material  which  had  been  made  stable  so  far  as  the  vital  influences 
of  the  haemolysis  were  concerned,  without,  however,  losing  its  chemi- 
cal character.  For  this  purpose  we  used  blood-cell  stromata,  by 


THE  POISON   OF  THE  COMMON  GARDEN  SPIDER.  171 

which  we  mean  blood-cells  deprived  of  their  haemoglobin  by  swell- 
ing and  then  again  condensing  the  blood-cell  residues.  Ehrlich1 
had  already  (in  1885)  pointed  out  the  importance  of  this  true  pro- 
toplasm of  the  blood-cells,  and  had  termed  it "  discoplasma  "  because 
of  its  peculiar  character.  According  to  Ehrlich,  the  main  function 
of  this  discoplasma  is  to  prevent  the  escape  of  the  haemoglobin,  and 
he  therefore  ascribed  the  diffusion  of  the  blood  coloring-matter  to 
death  of  the  discoplasma.  In  agreement  with  this  is  the  fact  first 
described  by  Bordet2  and  afterward  confirmed  by  Nolf,3  that  it 
is  the  stromata  which  bind  the  specific  serum  hsemolysins.  We 
could  therefore  assume  that  in  our  case,  in  all  probability,  the  arach- 
nolysin  would  be  bound,  if  bound  at  all,  by  the  stromata. 

In  this  Institute  a  method  for  the  production  of  the  stromata, 
which  differs  somewhat  from  the  one  commonly  employed,  has  proven 
particularly  valuable,  especially  in  studying  the  receptors.  With 
the  usual  solution  of  the  blood  in  distilled  water,  the  separation  by 
centrifuge  of  the  stromata  condensed  with  salt  is  extremely  dif- 
ficult; and  even  with  suitable  species  of  blood  only  a  small  yield  is 
obtained.  By  previously  heating  the  blood  we  have  found  that 
the  subsequent  centrifugation  is  made  considerably  easier  (perhaps 
because  of  a  kind  of  coagulation  of  the  blood-cells)  and  that  a  plentiful 
sediment  of  stromata  is  thereby  assured. 

The  blood  employed  is  heated  on  a  water-bath  at  50°-60°  C.  for  half  an 
hour  (depending  on  the  species  of  blood,  ox  blood  60°  C.,  rabbit  and  guinea- 
pig.blood  about  54°  C.)  until,  dark  brown  in  color,  it  just  begins  to  become  laky. 
Thereupon  the  blood,  made  up  to  6  to  10  volumes  by  the  addition  of  water  and 
shaken,,  is  mixed  with  so  much  salt  that  this  amounts  to  1%  of  the  total  amount. 
The  mixture  is  then  strongly  centrifuged.  The  stromata  remain  at  the  bottom 
of  the  vessel  in  the  form  of  yellowish-white  masses,  and  can  be  washed  by 
repeatedly  adding  NaCl  solution  and  centrifuging. 

The  stromata  so  obtained  have  preserved  their  receptor  property; 
they  bind  specific  serum  hcemolysins,  and  also,  when  introduced  into 
the  organism,  excite  the  production  of  specific  hosmolytic  immune  bodies* 

1  Ehrlich,  Zur  Physiologic  und  Pathologic  der  Blutscheiben,  Charit£  Annalen, 
X,  1885. 

2  Bordet,    Les    Serums    hemolytiques,    etc.,    Annales   de   PInstit.    Pasteur, 
1900. 

3  Nolf,  Le  Mecanisme  de  la  globulyse,  Annal.  de  PInst.  Pasteur,  1900. 

4  It  may  be  recalled  that  immunization  with  heated  bacteria  has  been  suc- 
cessfully practiced  even  from  the  beginning  of  the  study  of  immunity. 


172  COLLECTED   STUDIES  IN  IMMUNITY. 

The  fact  that  they  have  suffered  a  certain  quantitative  loss  in 
these  properties,  owing  to  the  extensive  manipulation  to  which  they 
have  been  subjected,  in  no  way  affects  their  utility  for  combining  ex- 
periments. In  the  qualitative  demonstration  of  specific  affinity  the 
employment  of  an  excess  of  receptors  answers  all  requirements. 

In  order,  furthermore,  to  meet  the  objection  of  a  mechanical  absorp- 
tion of  the  poison  by  the  stromata,  exactly  similar  combining  experi- 
ments were  made  simultaneously  with  a  blood  of  the  sensitive  class, 
and  with  one  of  the  insensitive  class.  As  a  representative  of  the 
former,  rabbit  blood,  which  is  highly  sensitive,  was  used.  For  the 
control,  guinea-pig  blood,  which  is  not  dissolved  by  arachnolysin, 
was  used.  The  degree  of  activity  of  the  poison  solution  before  and 
after  binding  was  measured  by  means  of  rabbit  blood. 

The  stromata  sediments  derived  from  each  of  40  cc.  rabbit  blood  and  guinea- 
pig  blood,  are  mixed  each  with  10  cc.  of  an  arachnolysin  solution  of  which  0.025 
cc.  suffice  to  just  completely  dissolve  0.05  cc.  rabbit  blood.  The  stromata  so 
treated  are  digested  for  half  an  hour  in  the  water-bath  at  40°  C.,  being  re- 
peatedly shaken.  They  are  theti  centrifuged.  The  decanted  fluid  from  the 
stromata  of  guinea-pig  blood,  like  the  original  material,  still  completely  dis- 
solves 0.05  cc.  rabbit-blood  in  amounts  of  0.025  cc.;  the  decanted  fluid  from 
the  rabbit  blood  stromata,  on  the  other  hand,  has  entirely  lost  its  poisonous 
action.  Even  in  amounts  of  1.0  cc.  it  is  unable  to  exert  the  least  action  on 
rabbit  blood. 

Hence  the  stromata  obtained  from  the  sensitive  blood  have  actually 
bound  the  arachnolysin,  and  this  combination  must  be  regarded  as  a 
chemical  one  because  the  control  test  with  guinea-pig  blood  shows 
that  the  insensitive  cell-material  exerts  no  attraction  whatever  on 
the  arachnolysin.  Such  behavior,  however,  is  most  easily  explained 
by  assuming,  in  accordance  with  the  side-chain  theory,  the  presence 
of  appropriate  receptors  in  the  sensitive  cells  as  a  prerequisite  for 
the  action  of  the  arachnolysin.  The  natural  immunity  of  certain 
species  of  blood  will  then  correspond  to  an  absence  of  appropriate 
receptors.  We  see  from  this  that  the  distribution  of  receptors  capable 
of  binding  arachnolysin,  at  least  so  far  as  the  blood  is  concerned,  is 
not  universal  throughout  the  animal  kingdom,  but  confined  to  certain 
species. 

While  the  experiences  already  mentioned  lead  us  to  regard 
arachnolysin  as  a  poison  belonging  to  the  class  of  toxins,  the  evidence 
will  be  made  absolutely  conclusive  by  demonstrating  the  ability  of  the 
poison  to  produce  antitoxin,  the  most  important  criterion  for  the 


THE  POISON  OF  THE  COMMON  GARDEN   SPIDER.  173 

toxin  nature  of  any  substance.  Owing  to  the  scarcity  of  material 
the  immunizing  experiments  were  somewhat  delayed;  they  will, 
however,  be  dealt  with  in  detail  at  the  proper  time.  Nevertheless 
I  can  announce  that  shortly  before  the  conclusion  of  this  work  we 
succeeded,  by  means  of  a  short  immunization  of  guinea-pigs  l  with 
garden-spider  poison,  to  produce  a  high-grade  antitoxic  serum,  of 
which  0.0025  cc.  sufficed  to  fully  protect  0.05  cc.  rabbit  blood  against 
a  complete  solvent  dose.  This  proves  the  toxin  nature  of  arachnolysin. 
In  conclusion  I  should  like  to  refer  to  the  relations  which  arachnoly- 
sin bears  to  what  we  know  about  spider  poisons  in  general.  In  doing 
so  I  shall  follow  Robert,2  who  made  the  fundamental  studies  in  the 
toxicology  of  animal  and  vegetable  poisons,  and  to  whom  we  owe 
most  of  our  knowledge  concerning  spider  poisons.  In  addition  to 
the  true  secretion  of  the  poison  gland,  Robert  distinguishes  "a  toxal- 
bumin  which  permeates  the  entire  body  of  the  spider,  even  the  legs 
and  eggs,  but  which  bears  no  necessary  relation  to  the  poison  gland." 
In  some  species  of  spiders  this  substance  mixes  with  the  gland  poison. 
According  to  Robert,  the  more  toxalbumin  gets  into  the  wound,  the 
stronger  are  the  constitutional  symptoms;  the  more  true  gland  poison, 
the  stronger  the  local  changes.  The  latter  is  especially  the  case  in  the 
lathrodectes  species  (malmignatte,  karakurte)  whose  sting  produces 
most  fearful  general  symptoms,  even  being  able  to  kill  human  beings. 
In  these  the  gland  secretion  becomes  dangerous  only  when  mixed 
with  toxalbumin  derived  from  the  body.  In  contrast  to  this,  the 
sting  of  the  garden  spider  produces  only  local  symptoms  of  irritation, 
although  the  spider's  body  contains  a  toxalbumin  whose  action  is 
analogous  to  the  preceding;  but  this  substance  does  not  become 
mixed  with  the  gland  secretion.  This  being  the  case,  it  is  very  likely 
that  the  haemolysin  described  by  us  is  identical  with  the  toxalbumin 
already  known  to  Robert;  for  we  also  obtained  it  from  the  body 
substance  of  the  garden  spider,  and  found  its  properties  to  be  those 
of  the  toxin. 

ADDITION  ON  REVISION. — Since  sending  in  the  manuscript  of  this  study  I 
have  learned  of  a  monograph  by  Robert  (Beitrage  zur  Kenntniss  der  Giftspinnen, 
Stuttgart,  1901)  which  has  just  appeared.  In  this  Robert  also  reports  on  the 
h.Tmolytic  action  of  the  poison  of  Rarakurtes  and  of  garden  spiders.  He  states 

1  Hence  although  guinea-pig  blood  is  insensitive  to  arachnolysin,  appropriate 
receptors  capable  of  binding  the  poison  must  be  present  in  the  guinea-pig  organism 
outside  of  the  blood. 

'Robert,  Lehrbuch  der  Intoxicationen,  Stuttgart,  1893,  p.  329. 


174  COLLECTED  STUDIES  IN  IMMUNITY. 

that  although  he  found  the  haemolytic  action  to  be  present  in  the  latter,  "it 
was  much  less  than  that  of  Karakurtes  poison."  It  is  possible,  however, 
that  Kobert  made  these  experiments  on  one  of  the  species  of  blood  found  by 
us  to  be  insensitive  to  arachnolysin  (horse  blood,  dog  blood?).  At  any  rate 
our  garden-spider  extract  far  exceeds  in  hsemolytic  action  the  Karakurtes  poison 
tested  by  Kobert  in  this  respect.  I  should  also  like  to  point  out  that  for  the 
haemolytic  experiments  with  Karakurtes  poison,  Kobert  used  dog  blood,  which 
according  to  our  table  belongs  to  the  class  of  blood  species  immune  to  garden- 
spider  poison.  Perhaps  in  conformity  with  the  extensive  analogy  between 
these  two  spider  poisons,  the  Karakurtes  poison  possesses  a  far  greater  haemo- 
lytic action  on  other  species  of  blood.  Kobert' s  observation  that  a  tolerance 
can  be  established  against  Karakurtes  poison  as  well  as  against  garden-spider 
poison,  agrees  very  well  with  the  idea  of  a  strong  antitoxic  serum,  a  fact  actually 
observed  by  us.  Since  then  we  have  obtained  such  a  serum  also  in  rabbits. 


XVI.   A  STUDY  OF  TOAD  POISON.* 

By  Dr.  FR.  PROSCHER. 

THE  numerous  investigations  concerning  toad  poison  which  have 
been  made  especially  by  French  and  Italian  workers,  have  not  yet 
come  to  a  definite  conclusion  as  to  whether  this  substance  is  alkaloid- 
like  or  toxin-like.  The  skin  secretions  of  the  different  varieties  of 
toads  contain  a  number  of  bodies  which  have  not  thus  far  been  studied. 
In  the  garlic  toad,  for  example,  there  is  a  substance  of  garlicky  odor, 
which  has  not  been  more  closely  identified.  Besides  this,  according 
to  Calmels,  toad  secretion  contains  methylcarby  laminic  acid  and 
methylcarbylamin,  which  are  said  to  act  intensely  on  the  nervous 
system.  Kobert  applied  the  name  "phrynin"  to  a  substance  wThiot 
irritates  the  mucous  membranes  very  intensely.  Phisalix  and 
Bertrand  claim  to  have  isolated  an  alkaloid  from  the  blood  serum 
of  the  common  toad,  but  it  remains  doubtful  whether  the  substance 
was  not  a  toxin,  for  they  wTere  unable  to  produce  it  in  chemically 
pure  form.  At  the  conclusion  of  their  investigations  they  themselves 
say  that  the  poisonous  action  is  not  due  entirely  to  the  "alka- 
loid." In  like  manner  Jornara  and  Casali  claim  to  have  isolated 
"bufidin"  from  dried  toad  poison.  They  say  .that  this  forms  crystal- 
line salts  and  must  therefore  be  an  alkaloid.  The  alcoholic  extract 
of  toad  skin  is  said  to  have  an  action  similar  to  digitalis.  Pugliese 
found  that  toad  poison  changes  haemoglobin  into  methaemoglobin, 
and  that  it  also  dissolves  the  blood-cells  outside  the  body.  Pugliese 
has  not  attempted  any  more  detailed  investigation.  From  the 
abstracts  of  his  study  at  my  disposal  I  was  unable  to  determine  the 
species  of  toad  used  in  his  experiments. 

The  object  of  the  following  investigation  is  to  furnish  a  small 
contribution  to  our  knowledge  of  toad  poison.  At  present  there 
can  be  no  thought  of  any  exact  analysis  of  the  poison. 

1  Reprint  from  Beitrage  zur  chemischen  Physiologie  u.  Pathologie,  Vol.  1, 
Nos.  10-12. 

175 


176  COLLECTED  STUDIES  IN   IMMUNITY. 

METHOD    OF   OBTAINING   THE    POISON. 

The  toad  poison  used  in  my  experiments  was  derived  from 
bombinator  igneus,  the  fire-toad,  and  from  bufo  cinereus,  the  common 
garden  toad.  In  order  to  obtain  the  poison,  the  skin  of  the  abdomen 
and  back  of  a  freshly  captured  toad  was  used,  for  the  poison  is  present 
in  largest  amounts  in  the  skin.  The  muscles  and  blood  serum  of 
the  fire- toad  also  contain  the  poison,  but  in  smaller  quantities. 

After  the  toads  were  thoroughly  rinsed  with  physiological  salt 
solution  they  were  decapitated  and  skinned.  The  skin  was  again 
rinsed  with  salt  solution  and  then  rubbed  to  a  paste,  as  homogeneous 
as  possible,  with  powdered  glass.  After  adding  2  to  3  cc.  physiological 
salt  solution  the  mixture  was  filtered  or  centrifuged.  The  resulting 
fluid  had  a  feebly  acid  reaction,  a  greyish  white  color  and  a  peculiar, 
garlicky  odor.  Toluol  was  added  as  a  preservative,  and  the  fluid 
'stored  in  the  refrigerator.  In  the  same  manner  I  prepared  an  extract 
from  the  skin  of  the  garden  toad. 

The  extract  of  the  skin  of  the  fire-toad  showed  strong  hsemolytic 
properties;  that  of  the  garden  toad  the  same,  though  only  in  traces. 
(See  Table  III.)  The  following  experiments  refer  only  to  the  fire- 
toad  poison  which,  for  short,  we  shall  call  "phrynolysin."  The  poison 
of  the  garden  toad  was  used  merely  for  comparison. 

PROPERTIES    OF    PHRYNOLYSIN. 

Phrynolysin  is  an  exceedingly  labile  body.  Heating  to  56°  C., 
exposure  to  light,  the  addition  of  alcohol,  ether,  chloroform,  min- 
eral acids,  strong  potash  lye,  pepsin  and  trypsin,  all  destroy  it  in  a 
short  time.  Drying  the  'phrynolysin  over  anhydrous  phosphoric  acid 
at  room  temperature  weakens  it  materially.  It  does  not  dialyze. 

Since,  as  already  mentioned,  the  extract  from  the  toad  skin  pos- 
sesses a  faint  acid  reaction,  requiring  1  to  1.3  cc.  decinormal  lye 
for  neutralization,  it  could  be  assumed  that  the  acid  reaction  slowly 
destroys  the  toxin.  The  destruction  of  the  toxin,  however,  proceeds 
in  the  same  time  in  neutral  as  in  feebly  acid  solution,  so  that  the 
acid  reaction  cannot  possess  any  great  influence.  The  hsemolytic 
action  is  the  same  in  acid  as  in  neutral  solution. 

The  best  preservative  for  this  substance  is  toluol,  first  employed 
by  Ehrlich  for  preserving  the  toxins.  Cold  storage  is  also  good. 
After  a  time  the  fluid  becomes  cloudy,  owing  to  the  separation  of 
albumin,  but  it  maintains  its  hffimolytic  power  unimpaired  for  A 


A  STUDT  OF  TOAD  POISON.  177 

considerable  time.  After  from  one  to  two  months  the  phrynolysin 
gradually  becomes  inert.  Owing  to  the  extreme  lability  of  the  toxin 
there  can,  for  the  present,  be  no  thought  of  obtaining  the  substance 
pure,  for  even  drying  at  room  temperature  weakens  the  poison  con- 
siderably. Owing  to  lack  of  material,  a  pharmacological  examination 
of  the  poison  could  not  be  undertaken. 

BEHAVIOR    OF   THE    PHRYNOLYSIN   TOWARD    DIFFERENT   SPECIES   OF 

BLOOD. 

The  method  of  testing  was  such  that  a  series  of  test-tubes  was 
prepared,  each  containing  1  cc.  of  the  dilution  1:10,  1:20,  etc.,  i.e., 
decreasing  amounts  of  the  poison.  The  dilutions  were  made  with 
0.85%  salt  solution.  To  each  tube  1  cc.  of  the  5%  blood  suspension 
in  0.85%  salt  solution  was  added.  Thereupon  the  tubes  were  kept 
at  37°  C.  for  two  hours  and  in  the  refrigerator  overnight.  A  "com- 
plete solution  "  is  one  that  on  shaking  shows  no  body  elements  of 
any  kind:  "almost  complete  "  if  there  is  still  a  slight  sediment;  and 
"  incomplete  "  when  numerous  blood-cells  are  undissolved.  This  is 
followed  in  order  by  "red,"  "top,"  "trace,"  "O." 

Commencing  with  Table  III  all  the  experiments  are  made  on  sheep 
blood. 

As  can  be  seen  from  Table  I,  sheep  blood  is  most  strongly  dis- 
solved, frog  and  toad  blood  not  at  all.  The  limits  of  solution  for 
sheep  blood  are  a  dilution  of  1:10240  in  the  case  of  phrynolysins 
I  and  II,  and  1:5120  in  phrynolysin  III.  In  Table  IV,  decreasing 
amounts  of  the  poison  are  added  to  1  cc.  5%  sheep  blood.  Of  phry- 
nolysin I,  0.0025  cc.  sufficed  to  effect  complete  solution;  of  II  and 
III,  0.00025  cc.  sufficed,  and  of  IV,  0.005  *cc.  By  determining  the 
amount  of  dry  residue  hi  poison  solution  II  it  is  seen  that  0.0000022 
g.  of  organic  substance  suffice  to  completely  dissolve  1  cc.  5%  sheep 
blood.  Of  poison  solution  III,  0.0000015  g.  have  the  same  effect. 
If  we  assume  that  one-tenth  of  this  organic  substance  (probably 
it  is  still  less)  represents  true  phrynolysin,  the  rest  being  merely 
indifferent  albuminous  bodies,  we  find  that  3/io  mg.  are  sufficient 
to  completely  dissolve  one  liter  of  sheep  blood. 

The  yield  of  phrynolysin  is  subject  to  individual  fluctuations. 
Animals  freshly  caught  yield  a  stronger  haemolysin  than  those  which 
have  been  kept  in  captivity  for  some  time. 


178 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE  I. 


Dilution. 

Sheep  Blood. 

Goat  Blood. 

Rabbit  Blood. 

Dog  Blood. 

Ox  Blood. 

1:20 

complete 

complete 

complete 

complete 

top 

1:40 

1:80 

(i 

t 

red 

1  1 

1:160 

« 

t 

top 

trace 

1:320 

« 

red 

« 

0 

1:640 

n 

' 

tt 

0 

1:1280 

it 

' 

if 

0 

1:2560 

incomplete 

trace 

K 

0 

1:5120 

almost 
complete 

top 

0 

0 

0 

1:10240 

red 

trace 

0 

0 

0 

1:20480 

trace 

0 

0 

0 

0 

1:40960 

0 

0 

0 

0 

0 

Dilution. 

Chicken  Blood. 

Guinea-pig  Blood. 

Rat  Blood. 

Pigeon  Blood. 

1:20 

incomplete 

red 

red 

trace 

1:40 

red 

" 

top 

0 

1:80 

« 

top 

(i 

0 

1:160 

0 

trace 

trace 

0 

1:320 

0 

0 

0 

0 

Dilution. 

Pigeon  Blood. 

Goose  Blood. 

Frog  Blood. 

Toad  Blood. 

1:20 

trace 

red 

0 

0 

1:40 

0 

top 

0 

0 

1:80 

0 

0 

0 

0 

TABLE  II. 
PHRYNOLYSIN  OF  THE  COMMON  GARDEN  TOAD. 


Dilution. 

Sheep  Blood. 

Goat  Blood. 

Dog  Blood. 

Rabbit 
Blood. 

Guinea-pig 
Blood. 

Ox  Blood. 

1:20 

red 

0 

red 

0 

0 

0 

1:40 

trace 

0 

top 

0 

0 

0 

1:80 

a 

0 

« 

0 

0 

0 

TABLE  III. 
BEHAVIOR  OF  DIFFERENT  PHRYNOLYSINS  TOWARD  SHEEP  BLOOD. 


Dilution. 

Phrynolysin  I. 

Phrynolysin  II. 

Phrynolysin  III. 

1:640 

complete 

complete 

complete 

1:1280 

1:2560 

« 

tt 

(  I 

1:5120 

tt 

ft 

almost  complete 

1:10240 
1:20480 

almost  complete 
top 

almost  complete 
red 

£5 

A  STUDY  OF  TOAD  POISON. 


179 


TABLE  IV. 
BEHAVIOR  OF  DIFFERENT  PHRYNOLYSINS  TOWARD  SHEEP  BLOOD. 


cc. 

Phrynolysin  I. 

Phrynolysin  II. 

Phrynolysin  III. 

Phrynolysin  IV. 

0.005 

complete 

complete 

complete 

complete 

0.0025 

incomplete 

0.001 

incomplete 

top 

0.00075 

red 

« 

0 

0.0005 

top 

(( 

0 

0.00025 

0 

0 

0.0001 

0 

incomplete 

red 

0 

ATTEMPTS  AT  REACTIVATING  A  PHRYNOLYSIN  WHICH  HAD   BECOME 
INACTIVE    AT   56°  C. 

The  investigations  of  Ehrlich  and  Morgenroth  have  shown  that 
the  hsemolysins  of  the  higher  vertebrates  are  of  complex  constitu- 
tion. They  consist  of  two  portions,  the  complement  and  the  immune 
body.  By  heating  to  56°  C.  the  complement  is  destroyed,  while 
the  immune  body  remains  intact.  The  immune  body  by  itself  can- 
not exert  any  hsemolytic  action;  a  fitting  complement  must  first 
be  added. 

It  would  be  quite  comprehensible  for  the  phrynolysin  likewise 
to  consist  of  two  parts.  Heating  to  56°  C.  would  destroy  the  com- 
plement, while  the  thermostable  interbody  would  be  preserved. 
I  therefore  attempted  to  reactivate  the  toxin  which  had  become 
inactive  at  56°  C.  and  tried  the  addition  of  a  number  of  different 
normal  serum  for  this  purpose,  such  as  goat  serum,  sheep  serum, 
pigeon  serum,  horse  serum,  guinea-pig  serum,  and  rabbit  serum, 
all  without  success,  no  solution  taking  place.  Unfortunately,  owing 
to  lack  of  material,  I  was  unable  to  obtain  1  or  2  cc.  of  serum  from 
the  fire-toad  in  order  to  employ  this  for  reactivation.  Experiments 
with  the  normal  sera  of  the  higher  vertebrates  are  not  conclusive, 
because  the  complement  sought  for  may  possibly  be  contained  only 
in  the  serum  of  the  fire- toad.  For  the  present  therefore  the  ques- 
tion as  to  the  complex  character  of  the  phrynolysin  must  still  be 
kept  open. 

DO    NORMAL    SERA    CONTAIN    ANTIBODIES    AGAINST    PHRYNOLYSIN? 

A  number  of  normal  sera,  which  had  first  been  inactived  at  56°  C. 
in  order  to  avoid  solution  of  the  sheep  blood  added,  were  tested  for 
this  purpose,  e.g.,  pigeon  serum,  sheep  serum,  guinea-pig  serum, 
horse  serum,  rabbit  serum,  and  goat  serum.  None  of  these  sera, 


180  COLLECTED   STUDIES  IN  IMMUNITY. 

even  in  amounts  up  to  1  or  2  cc.  was  able  to  prevent  solution,  although 
only  the  single  solvent  dose  of  phrynolysin  was  added  to  the  mix- 
ture of  blood  and  serum. 

IMMUNIZATION   WITH   PHRYNOLYSIN. 

In  order  to  furnish  conclusive  proof  that  phrynolysin  is  a  true 
toxin,  a  number  of  rabbits  were  immunized  with  the  same.  The 
poison  was  injected  subcutaneously,  commencing  with  \  cc.  and 
increasing  to  5  cc.  in  the  course  of  eight  days.  The  dose  of  5  cc. 
was  then  injected  every  5  to  6  days  for  two  or  three  times  so  that 
in  the  course  of  three  weeks  about  30-35  cc.  had  been  given. 

It  is  not  advisable  to  give  more  than  5  cc.  at  once  because  other- 
wise the  animals  die  in  one  or  two  days.  The  anatomical  findings 
in  an  animal  which  has  died  of  toad  poison  are  negative,  excepting 
a  marked  hypersemia  of  the  abdominal  viscera;  no  macroscopical 
changes  of  the  organs  are  demonstrable. 

As  already  mentioned,  normal  rabbit  serum  does  not  contain 
a  trace  of  anti-body  against  phrynolysin.  The  production  of  anti- 
toxin commences  about  fourteen  days  after  the  injection  of  the  toxin 
and  reaches  its  maximum  in  three  weeks.  Of  the  strongest  serum 
which  I  obtained,  0.025  cc.  protected  against  double  the  solvent 
dose  of  phrynolysin  for  1  cc.  5%  sheep  blood. 

LITERATURE. 

VULPIAN,  Comp.  rend,  de  la  soc.  de  biol.,  1854,  p.  133;  1856,  p.  124. 

DOM.  JORNARA,  Sur  les  effets  physiol.  du  venin  de  crapaud.,  Journ.  de  Therap., 

4,  p.  833  et  929. 

G.  CALMELS,  Comp.  rend.,  98,  536  (1883),  and  Archiv.  de  physiol.,  1883. 
CAPPARELLI,  Archiv.  ital.  de  biol.,  4,  72  (1883). 
KOBERT,  Sitzungs-Bericht   der  Dorpater  Naturforschenden  Gesellschaft,  9,  63 

(1891). 
PHISALIX  and  G.  BERTRAND,  Toxische  Wirkung  von  Blut  und  Gift  der  gemeinen 

Krote,  Comp.  rend.,  116,  1080-1082,  Arch,  de  physiol.,  25,  511,  and  517, 

Comp.  rend.  soc.  biolog.,  45,,  477,  479. 
D.  JORNARA  and  CASALI,  Das  Gift  der  Krote  und  das  Bufidin,  Revista  clinica 

Bologne,  1873. 
A.  PUGLIESE,  Die  Methsemoglobinbildende  Wirkung  des  Krotengiftes,  Archiv. 

di  farmac.  e  terap.,  1898. 
EHRLICH  and  MORGENROTH,  Uber  Haemolysine,  Berl.  klin.  Wochenschr.    1899, 

Nos.  1  and  22;  1900,  Nos.  21  and  31;  1901,  Nos.  10,  21,  and  22. 


XVII.   CONCERNING  ALEXIN  ACTION.* 

By  Dr.  HANS  SACHS,  Assistant  at  the  Institute. 

AFTER  the  fundamental  studies  of  Bordet,  and  of  Ehrlich  & 
Morgenroth  had  shown  that  the  hsemolysins  produced  in  serum  by 
immunization  with  blood-cells  owed  their  effect  to  the  combined 
action  of  two  substances  (amboceptor  and  complement)  it  seemed 
very  natural  to  suppose  that  the  haemolysins  of  normal  sera,  which 
had  been  known  for  some  time,  were  also  of  a  complex  nature. 
Buchner,  who  was  the  first  to  recognize  the  significance  of  the  bac- 
tericidal and  globulicidal  properties  of  blood  serum,  had  conceived 
these  actions  from  a  Unitarian  standpoint  and  referred  them  to  the 
"  alexin  "  of  each  particular  serum.  Recent  investigations,  how- 
ever, have  shown  that  Buchner 's  alexin  is  not  a  single  simple  sub- 
stance, but  the  sum  of  an  infinite  number  of  combinations,  whose  more 
thorough  analysis  has  been  rendered  possible  only  by  the  methods 
of  the  newer  hsemolysin  investigations. 

The  credit  of  applying  the  experiences  gained  with  the  hsemoly- 
sins artificially  produced  to  the  study  of  hsemolysins  of  normal 
serum,  belongs  to  Ehrlich  and  Morgenroth.  They  made  use  of  a 
method  which  they  had  already  employed  in  the  analysis  of  haemolytic 
immune  sera,  the  separation  by  means  of  cold.  This  depends  on  the 
fact  that  at  0°  under  favorable  circumstances  only  the  interbody, 
and  not  the  complement,  is  bound  by  the  blood-cells.  Accordingly 
by  appropriate  treatment  it  could  be  shown  that  the  serum  had 
lost  part  of  its  power,  but  that  it  could  be  regenerated  by  the  addi- 
tion of  the  same  kind  of  serum  previously  inactivated  by  heat.  This 
confirmed  their  view  of  the  complex  nature  of  normal  hsemolysins. 
They  were  further  able  to  activate  inactive  hsemolytic  normal  sera 
by  the  addition  of  other  kinds  of  sera  which  served  as  complements, 
and  which  by  themselves  did  not  dissolve  the  particular  blood-cells 

1  Reprint  from  the  Berl.  klin.  Wochen.  1902,  Nos.  9  and  10. 

181 


182  COLLECTED  STUDIES 'IN  IMMUNITY. 

used.  This  showed  conclusively  that  the  globulicidal  yroperty  of 
normal  serum  is  due  to  the  co-action  of  two  bodies,  one  which  withstands 
heating  (thermostable]  and  the  other  which  does  not  (thermolabile .) 

These  views  have  been  accepted  by  the  majority  of  investiga- 
tors, and  numerous  observers,  P.  Miiller,1  London,2  E.  Neisser, 
and  Doring3  have  constantly  added  new  facts,  the  analysis  of 
which  in  every  instance  demonstrates  the  complex  nature  of  nor- 
mal hsemolysins.  Nevertheless  it  does  not  seem  to  me  superfluous 
to  thoroughly  discuss  this  question  once  more,  since  such  eminent 
authorities  as  Buchner  4  and  Gruber,5  because  of  the  negative  result 
of  part  of  their  experiments,  hold  that  Ehrlich  and  Morgenroth's 
conception  of  the  nature  of  normal  hsemolysins  is  erroneous.  Ehrlich 
and  Morgenroth  had  from  the  beginning  stated  that  the  solution  of 
this  problem  in  any  particular  case  was  only  possible  by  their  method 
under  certain  favorable  circumstances.  Now,  although  Buchner  and 
Gruber  have  employed  this  method,  so  that  a  negative  result  proves 
nothing  whatever,  in  consideration  of  the  importance  of  the  matter 
I  have  followed  the  suggestion  of  Prof.  Ehrlich,6  and  undertaken 
a  critical  study  of  the  negative  findings  of  these  authors.  The  results 
of  this  have  already  been  briefly  alluded  to  elsewhere. 

Buchner  sought  to  discover  the  presence  of  thermostabile  bodies 
(his  "  Hilfskorper  ")  on  the  occurrence  of  haemolysis,  by  reactivat- 
ing normal  sera,  which  had  been  inactivated  by  heating  to  60°  Cv 
with  fresh  serum  of  a  different  species.  But  out  of  the  large  number 
of  possible  combinations  he  chose  only  one  and  used  as  a  source  of 
complement  only  that  serum  which  was  derived  from  the  same  species 
that  furnished  the  blood-cells.  In  an  address  on  the  protective 
bodies  of  the  blood,  delivered  at  the  Hamburg  Congress  of  Natu- 
ralists, Ehrlich  pointed  out  that  this  procedure  was  inapplicable. 
It  can  surely  not  be  expected  that  every  serum  contains  a  fitting 

1  P.  Miiller,  Uber   Antihamolysine,  Centralblatt  fiir  Bacteriologie,  Vol.  29, 
1901. 

2  E.  S.  London,  Contribution  a  l'4tude  des  h&nolysines,  Arch,  des  Sciences 
biolog.  (Inst.  imperial  de  me*d.  exper.  a  St.  Petersbourg),  T.  VIII,  1901. 

3  E.  Neisser  u.  Doring,  Zur  Kenntniss  der  hamolytischen  Eigenschaften  des 
menschlichen  Serums,  Berl.  klin.  Wochen.  1901,  No.  22. 

4  Buchner,   Sind  die  Alexine  einfache  oder  complexe  Korper?     Berl.  klin. 
Wochenschr.  1901,  No,  33. 

5  M.  Gruber,  Zur  Theorie  der  Antikorper,  II,  Uber  Bacteriolyse  u.  Haemolyse, 
Munch,  med.  Wochenschr.  1901,  48  and  49, 

8  Ehrlich,  Vortrag  im  Verein  fur  innere  Medicin,  Dec,  16,  1901. 


CONCERNING  ALEXIN  ACTION.  183 

complement  for  any  given  amboceptor.  In  testing  a  series  of  com- 
binations, therefore,  the  finding  of  a  suitable  complement  will  to  a 
certain  extent  be  merely  a  coincidence.  In  all  the  cases  studied 
at  this  Institute,  however,  even  though  often  only  after  consider- 
able labors,  this  has  always  led  to  a  certain  realization  of  the  com- 
plex nature  of  the  haemolysin. 

Buchner  was  successful  in  two  of  his  cases  in  activating  the  combi- 
nation chosen  by  him:  blood-cells  A  +  inactive  serum  (amboceptor)  B 
-\-active  serum  (complement)  A.  (Guinea-pig  blood  and  ox  serum; 
goat  blood  and  rabbit  serum.)  In  three  other  cases,  however,  he 
was  unable  with  a  corresponding  mode  of  procedure  to  restore  the 
solvent  power  which  had  been  lost  by  inactivation.  Guinea-pig 
blood  and  sheep  serum  (Case  I);  sheep  blood  and  rabbit  serum 
(Case  II);  guinea-pig  blood  and  dog  serum  (Case  III).  These  results, 
to  be  sure,  are  contrary  to  those  of  Ehrlich  and  Morgenroth,  who 
observed  more  or  less  marked  haemolysis  in  these  same  combina- 
tions. These  opposing  results  are,  however,  explained  first  by  the 
fact  that  the  amount  of  complement  contained  hi  the  serum  of  the 
same  species  is  subject  to  individual  and  chronological  variations 
within  wide  limits.  Beside  this,  recent  experiences,  which  we  shall 
subsequently  discuss  in  detail,  have  shown  us  that  the  temperature 
at  which  the  serum  is  inactivated  is  not  indifferent  for  the  function 
of  the  amboceptor.  Hence  it  appears  significant  that  in  these  experi- 
ments Buchner  inactivated  the  sera  by  heating  to  60°  C.,  whereas 
ordinarily  this  is  done  at  56°-57°  C.  As  a  matter  of  fact,  Buchner's 
experiment  No.  6  shows  that  dog  serum,  in  this  experiment  inac- 
tivated by  heating  only  to  57°  C.,  is  activated  hi  its  haemolytic  action 
for  guinea-pig  blood  by  rabbit  serum.  In  view  of  this,  the  nega- 
tive7 findings  of  Buchner  in  Case  III  lose  their  significance. 

In  the  three  cases  looked  upon  by  Buchner  as  negative,  I  tried, 
by  separation  by  means  of  cold,  to  convince  myself  of  the  presence 
of  two  substances  effecting  the  haemolysis.  My  method  of  pro- 
cedure was  as  follows: 

Two  parallel  series  of  tubes  of  blood  containing  decreasing  amounts 
of  active  serum  were  prepared,  kept  at  0°  C.  for  2-3  hours  and  then 
centrifuged.  The  decanted  fluid  of  one  series  was  then  allowed  to 
act  on  the  sediments  of  native  blood,  that  of  the  other  series  on  the 
sediments  of  blood  which  had  been  treated  with  a  like  quantity  of 
inactivated  serum.  The  amount  of  blood,  as  in  all  our  experiments, 
was  1  cc.  of  a  5%  suspension  in  .85%  salt  solution. 


184 


COLLECTED  STUDIES  IN  IMMUNITY. 


In  two  combinations  (Cases  I  and  II)  the  separation  of  the  two 
components  was  effected  without  any  trouble.  The  following  pro- 
tocol will  also  show  the  technique  of  the  experiment. 

Negative  case  I  of  Buchner.  0.5  cc.  sheep  serum  is  still  just 
able  to  completely  dissolve  guinea-pig  blood.  To  each  1  cc.  of  a 
5%  guinea-pig  blood  suspension  varying  amounts  of  active  sheep 
serum  are  added  and  the  volume  of  fluid  made  up  to  2  cc.  with  physio- 
logical salt  solution.  Two  parallel  series  like  this  are  kept  at  0°  C. 
for  two  hours  and  then  centrifuged.  The  clear  decanted  fluids  from 
the  one  series  are  allowed  to  act  each  on  the  sediment  of  1  cc.  native 
5%  guinea-pig  blood;  the  fluids  from  the  other  series,  each  on  the 
sediment  of  1  cc.  5%  guinea-pig  blood,  which  had  previously  been 
treated  with  the  same  varying  amounts  of  inactive  sheep  serum. 
The  hsemolytic  action  of  the  decanted  fluids  is  shown  by  Table  I. 

TABLE  I. 
ABSORPTION  OF  SHEEP  SERUM  BY  GUINEA-PIG  BLOOD  AT  0°  C. 


Amount  of  the 
Sheep  Serum 
Added. 

cc. 

Solvent  Power  of  the  Decanted  Fluids  for 

A,  Native 
Guinea-pig  Blood. 

B,  Guinea-pig  Blood 
Previously  Treated 
with  Inactive 
Sheep  Serum. 

1       0.7 
2      0.6 
3       0.5 
4      0.4 
5      0.35 
6      0 

moderate 

(  f 

little 

trace 
i  i 

0     • 

complete 

tt 

almost  complete 
strong 
0 

Buchner's  second  negative  case  deals  with  the  combination  sheep 
blood  and  rabbit  serum.  In  the  following  experiment,  entirely  ana- 
logous to  the  preceding,  the  complete  solvent  dose  of  rabbit  serum 
for  sheep  blood  was  0.2  cc.  See  Table  II. 

These  experiments,  which  are  confirmed  by  numerous  parallel 
experiments,  show  that  in  these  two  cases,  as  a  matter  of  fact,  hcemolysis 
depends  on  the  presence  of  two  substances.  One  of  these,  thermostable, 
is  bound  by  the  blood-cells  at  0°C.,  the  other,  thermolabile,  is  left 
behind  at  this  temperature.  The  latter,  however,  is  only  then  able 
to  effect  haemolysis  when  it  acts  on  blood-cells  which  have  previously 
anchored  the  thermostable  substance,  the  amboceptor. 

A  comparison  of  Tables  I  and  II  also  shows  how  much  the 
combining  relations  between  amboceptor  and  blood-cell  on  the  one 


CONCERNING  ALEXIN  ACTION. 


185 


hand,  and  amboceptor  and  complement,  on  the  other,  may  vary 
from  case  to  case.      Whereas  in  Case  II  the  decanted  fluids  were 

TABLE  II. 
ABSORPTION  OF  THE  RABBIT  SERUM  BY  SHEEP  BLOOD  AT  0°  C. 


Amount  of 
Rabbit  Serum 
Added. 

cc. 

Solvent  Power  of  the  Decanted  Fluids  for 

A,  Native 
Sheep  Blood. 

B.  Sheep  Blood 
Previously  Treated 
with  Inactive 
Rabbit  Serum. 

1       0.6 
2      0.45 
3      0.35 
4      0.25 
5      0.2 
6      0 

trace 
0 
0 
0 
0 
0 

complete 

almost  complete 

moderate 

<  < 

0 

absolutely  inactive  against  native  blood  (i.e.,  all  the  amboceptor 
had  been  bound  at  0°  C.  by  the  blood-cells)  in  Case  I  the  decanted 
fluids  were  then  still  active  when  the  amounts  of  serum  added  were 
less  than  the  solvent  dose.  This  indicates  that  in  this  case  the  affinity 
of  the  amboceptor's  cytophile  group  for  the  receptor  of  the  cell  is 
relatively  slight  at  0°  C.  In  like  manner  the  columns  B  of  the  tables 
show  a  certain  difference  of  affinity  between  amboceptor  and  com- 
plement. In  Case  I  the  decanted  fluid  still  contains  the  entire  com- 
plement; in  Case  II,  on  the  other  hand,  a  portion  of  the  complement 
must  have  combined  with  the  amboceptor,  for  the  decanted  fluid 
shows  a  distinct  loss  of  complement.  The  separate  examination  of 
the  sediments  of  the  specimens  to  which  active  serum  was  added 
agrees  with  this;  in  Case  I  these  sediments  mixed  with  physiological 
salt  solution  and  placed  into  the  incubator  showed  no  trace  of  solution, 
while  in  Case  II  the  sediments  of  the  first  three  tubes  showed  mod- 
erate, little,  and  trace  of  solution  respectively. 

Both  normal  hoemolysins  (Buchner's  negative  Cases  I  and  II)  therefore 
correspond  in  their  main  behavior.  They  consist  of  two  components 
(readily  separable  by  the  "cold  method")  which  in  their  mutual  relations 
manifest  a  certain  variation  in  the  behavior  of  their  receptors. 

The  conditions  in  these  two  combinations  were  favorable  for 
analysis  of  the  mode  of  action  by  means  of  our  method.  In  the 
study  of  Buchner's  third  negative  case,  however  (guinea-pig  blood 
and  dog  serum),  difficulties  presented  themselves  which  at  first  ap~ 


186 


COLLECTED  STUDIES  IN  IMMUNITY. 


peared  to  be  insurmountable.  Despite  numerous  variations  in  the 
conditions  of  the  experiment  we  did  not  succeed  with  appropriate 
procedures  in  effecting  a  separation  by  means  of  the  "cold  method. " 
The  fluids  decanted  from  the  mixture  of  guinea-pig  blood-cells  and 
active  dog  serum  manifested  the  same  behavior,  so  far  as  hamolytic 
action  was  concerned,  on  normal  guinea-pig  blood  and  such  as  had 
previously  been  treated  with  inactive  dog  blood;  and  yet  they  showed 
slight  differences  so  that  we  did  not  feel  justified  in  drawing  any 
conclusion.  However,  we  soon  became  convinced  that  a  separation  of 
two  substances  causing  haBmolysis  had  nevertheless  been  effected  by 
the  absorption  in  the  cold.  We  allowed  the  fluid  decanted  from  the 
guinea-pig  blood-cells  previously  treated  with  active  dog  serum,  which 
fluid  only  slightly  dissolved  native  guinea-pig  blood,  to  act  on  guinea- 
pig  blood  sediments,  which  also  had  previously  been  mixed  with 
active  dog  serum.  We  were  then  able  to  determine  that  these  sedi- 
ments were  strongly,  in  appropriate  quantities  completely,  dissolved 
by  the  decanted  fluid,  although  when  mixed  merely  with  salt  solution 
and  placed  into  an  incubator  they  did  not  dissolve  at  all,  or  dissolved 
only  in  traces.  An  experiment  of  this  kind  is  shown  in  Table  III. 

TABLE  III. 
ABSORPTION  OF  DOG  SERUM  BY  GUINEA-PIG  BLOOD  AT  0°  C. 


Solvent  Power  of  the  Decanted  Fluids  for 

Amount  of 
Dog  Serum 
Added. 

Haemolysis  of  the 
Sediments  Sus- 
pended in  Salt 
Solution  at  37°. 

A,  Native  Guinea- 

B,  Guinea-pig 
Blood  Previously 
Treated  with 

C,  Guinea-pig 
Blood  Previously 
Treated  with 

Inactive  Dog 

Active  Dog  Serum 

cc. 

Serum. 

at  0°. 

1       0.25 
2       0.2 

trace 
faint  trace 

almost  complete 
strong 

complete 
almost  complete 

complete 

3       0.15 

0 

moderate 

moderate 

4       0.1 

0 

little 

little 

(  e 

5      0.075 

0 

trace 

trace 

strong 

Hence  by  means  of  the  absorption  with  guinea-pig  blood  in  the  cold, 
the  active  dog  serum  was  separated  into  two  components  each  of  which 
by  itself  was  incapable  of  effecting  solution.  One  of  these  became  attached 
to  the  red  blood-cells,  the  other  remained  in  the  fluid.  The  former  there- 
fore corresponded  in  its  behavior  to  the  amboceptor,  and  it  was  only 
a  coincidence  that  dog  serum  inactivated  by  heating  to  60°  C.  was  unable 
also  to  assume  that  role.  We  hoped  to  discover  more  about  the  nature 
of  this  curious  behavior  by  employing  a  different  method  of  inactivat- 


CONCERNING  ALEXIN   ACTION. 


187 


ing  the  dog  serum,  and  therefore  in  this  case  turned  first  to  the  com- 
pletion method.  By  means  of  completion  of  the  variously  inactivated 
dog  serum  with  other  sera  which  do  not  dissolve  guinea-pig  blood, 
we  hoped  to  obtain  an  insight  into  the  circumstances  here  presented. 
In  this  way  we  were  able  to  convince  ourselves  that  dog  serum  which 
had  been  inactivated  by  half  an  hour's  heating  to  60°  C.  according  to 
Buchner's  procedure,  is  no  longer  activated  in  its  hsemolytic  action 
on  guinea-pig  blood,  by  the  addition  of  guinea-pig  serum.  When 
the  dog  serum,  however,  was  heated  only  to  55°  C.  or  even  only  to 
50°  C.  it  was  always  possible  to  activate  such  an  inactivated  serum 
by  means  of  guinea-pig  serum.  This  was  the  more  readily  effected, 
the  lower  the  inactivating  temperature  employed.  It  need  hardly 
be  mentioned  that  in  particular  cases  we  always  determined  whether 
the  serum  really  was  inactive;  and  this  showed  that  dog  serum 
loses  its  hsemolytic  property  for  guinea-pig  blood  completely,  even 
when  merely  warmed  for  half  an  hour  to  49°  C.  We  must  therefore 
regard  it  as  a  fortunate  coincidence  that  the  complement  of  dog 
serum  is  so  markedly  thermolabile,  for  only  under  this  condition  could 
it  be  possible  to  preserve  the  amboceptor  intact,  i.e.,  capable  of  react- 
ing, for  that  body  is  but  little  more  stable.  Whether  the  amboceptor 
heated  to  60°  has  been  damaged  in  its  cy tophile  or  complementophile 
affinity  is  still  undetermined.  One  could  perhaps  also  think  of  a 
blocking  of  the  complementophile  group  of  the  amboceptor  due  to  a 
binding  of  the  comp  ement  taking  place  at  the  higher  temperature. 
Be  this  as  it  may,  these  experiments  certainly  show  that  the  power 
of  a  dog  serum  (inactivated  at  a  suitable  temperature,  e.g.,  50°  C.)  to 
be  activated  by  guinea-pig  serum  is  lessened  by  heating  the  dog  serum 
to  55°  C.  and  destroyed  at  60°  C. 

Table  IV  shows  such  an  experiment: 

TABLE  IV. 

COMPLETION  (WITH  GUIXEA-PIG  SERUM)  OF  DOG  SERUM  INACTIVATED  AT 
DIFFERENT  TEMPERATURES. 


Amount  of  the 
Activated  Guinea- 
pig  Serum. 

cc. 

Degree  of  Solution  of  the  Guinea-pig  Blood  Mixed  with  0.15  cc.  Dog 
Serum  and  Inactivated  by  Half  an  Hour's  Heating  to 

A,  60°. 

B,  55°. 

C,  50°. 

1         0.5 
2         0.25 
3        O.-l 
4         0 

}     • 

moderate 
little 
trace 
0 

complete 
strong 
little 
0 

188 


COLLECTED  STUDIES   IN   IMMUNITY. 


We  now  repeated  the  experiment  of  separation  in  the  cold  by 
allowing  the  fluid  which  was  decanted  from  the  guinea-pig  blood-cells 
after  these  had  been  treated  with  active  dog  serum  at  0°  C.,  to  act 
on  guinea-pig  blood  sediments  previously  mixed  with  dog  serum. 
Our  results  were  in  accord  with  the  above  and  led  to  a  clear  under- 
standing of  our  previous  negative  findings.  See  Table  V. 

TABLE  V. 

ABSORPTION  OP  DOG  SERUM  BY  GUINEA-PIG  BLOOD  AT  0°  C. 
(0.075  cc,  dog  serum  just  completely  dissolves  1  cc.  5%  guinea-pig  blood.) 


Amount  of 
Dog  Serum 
Added. 

cc. 

Solvent  Power  of  the  Decanted  Fluids  on 

A,  Native 
Guinea-pig 
Blood. 

B,  Guinea-pig   Blood   Previously  Treated   with  Dog 
Serum  Inactivated  at 

I,  60°. 

II,  55°. 

III.  50°. 

1     0.15 
2     0.1 
3     0.075 

complete 
moderate 
little 

complete 
almost  complete 
moderate 

complete 

<  « 

strong 

complete 

<  < 

« 

In  this  case,  therefore,  we  have  demonstrated  a  thermolability 
of  the  amboceptor1  which  shows  itself  especially  in  the  activa- 
ting experiment  with  guinea-pig  complement,  but  also  in  that  with 
its  own  dog  (complement).  Only  through  this  thorough  analysis 
was  it  possible  to  furnish  for  Buchner's  third  negative  case  also 
positive  proof  of  the  complex  constitution  of  normal  hsemolysins. 

After    having    determined    that    certain    amboceptors    will    only 


1  It  is  therefore  not  at  all  permissible  to  define  the  two  components  of  the 
haemolysin,  as  Gruber  would  do  (Discussion  of  Gruber's  Address,  Wiener  Klin. 
Wochensch.  1901,  No.  50),  only  according  to  the  temperature,  and  to  say  that  at 
a  certain  degree  of  heat  the  amboceptor  remains  intact  while  the  complement 
does  not.  As  long  ago  as  their  second  communication  Ehrlich  and  Morgenroth 
described  a  thermostable  complement  of  goat  serum  which  remained  intact  at 
56°  C. ;  and  according  to  our  experiences  here  described  a  general  definition  of 
amboceptors  as  bodies  which  withstand  heating  to  55°  C.  is  absolutely  impos- 
sible. The  influence  of  temperature  on  amboceptor  and  complement  varies 
from  case  to  case.  Hence  that  these  two  factors  act  together  in  haemolysis  we 
know  only  from  this,  that  two  substances,  in  themselves  not  capable  of  causing 
solution,  when  combined,  effect  hcemolysis;  and  that  one  of  these  substances  (the 
complement)  can  never  alone  be  bound  by  the  blood-cells  but  always  only  through 
the  intervention  of  the  other  (the  amboceptor). 


CONCERNING  ALEXIN   ACTION.  189 

bear  slight  warming,  in  order  to  remain  capable  of  reacting,  we  had 
to  abandon  our  custom  of  inactivating  sera  by  simply  heating  them 
to  60°  C.  Thereafter  we  had  always  first  to  determine  the  minimal 
inactivating  temperature  for  each  individual  case.  The  limits  of 
temperature  can  usually  be  determined  accurately;  for  dog  serum 
it  is  49°  C.  We  have  also  tried  by  means  of  other  complements  to 
activate  dog  serum  inactivated  at  50°,  and  have  found  a  suitable 
complement  not  only  in  guinea-pig  serum  but  also  in  human  serum. 
In  this  case  also,  the  thermolability  of  the  amboceptor  showed  itself, 
for  heating  to  60°  C.  destroyed  the  reactivatibility.  In  two  cases, 
however,  the  power  to  reactivate  was  preserved  to  a  greater  or  less 
extent  even  after  heating  to  60°  C.  In  like  manner  dog  serum 
could  be  activated  by  the  complements  described  when  it  had  been 
deprived  of  its  solvent  power  by  other  means.  Thus  the  comple- 
ments of  dog  serum  were  absorbed  by  means  of  yeast,  and  by  means 
of  an  anticomplement  serum  (from  a  goat)  whose  normal  amboceptor 
for  guinea-pig  blood  had  been  removed  by  washing  with  guinea-pig 
blood.  The  dog  sera  so  inactivated  manifested  their  amboceptor 
properties  when  they  were  appropriately  activated. 

In  the  first  two  negative  cases  of  Buchner,  separation  in  the  cold 
had  shown  the  presence  of  amboceptors  in  the  sheep  and  rabbit  serum. 
I  now  sought  by  means  of  activating  experiments  to  find  fitting 
complements  for  these  amboceptors  in  other  sera.  Naturally,  after 
the  above  experiences,  it  was  necessary  here  also  to  first  determine 
the  minimal  inactivating  temperature.  For  sheep  serum  this  is 
50°  C.,  for  rabbit  serum  51°  C.  If  sheep  serum  is  inactivated  by 
half  an  hour's  heating  to  50°  C.,  it  is  easy  to  restore  the  hsemolytic 
action  on  guinea-pig  blood  (Buchner's  Case  I)  by  the  addition  of 
fresh  human  serum.  In  this  way  the  complex  nature  of  the  normal 
haemolysin  of  sheep  serum  can  be  demonstrated.  One  can  also  acti- 
vate with  guinea-pig  serum,  although  then  a  feebler  solvent  action 
is  obtained.  In  both  cases  the  thermolability  of  the  amboceptor 
is  readily  demonstrated;  for  by  heating  the  sheep  serum  to  60°  C. 
this  can  no  longer  be  activated,  or  only  in  very  much  less  degree.1 


1  in  addition  to  this  I  have  also  demonstrated  a  thermolability  of  the  am- 
boceptors of  goat  serum  which  are  activated  by  horse  serum  and  act  on 
rabbit  and  guinea-pig  blood.  Repeated  investigations  by  Dr.  Morgenroth 
have  shown  that  a  markedly  thermolabile  amboceptor  is  contained  also  in  horse 
serum.  This  amboceptor,  which  fits  guinea-pig  blood,  and  can  no  longer  be 


190  COLLECTED  STUDIES  IN   IMMUNITY 

The  combination,  sheep  blood  and  rabbit  serum  (Buchner's 
second  case)  presents  entirely  analogous  conditions.  Both,  guinea- 
pig  serum  and  human  serum,  the  latter  only  in  a  moderate  degree 
contain  a  complement  which  activates  the  amboceptor  of  rabbit, 
serum.  The  rabbit  amboceptor,  however,  is  evidently  of  more  stable 
constitution;  for  even  after  heating  to  60°,  its  solvent  power  can 
be  completely  restored.  I  can  therefore  confirm  the  facts  found 
by  Buchner  in  this  case,  namely  that  sheep  serum  is  incapable  of 
restoring  the  solvent  power  for  sheep  blood.  This,  however,  accord- 
ing to  the  above  statement,  is  naturally  no  argument  against  the- 
complex  nature  of  the  hsemo'ysin  because  not  every  serum  need  con- 
tain a  fitting  complement  for  every  particular  amboceptor. 

Provided  that  sufficiently  numerous  combinations  are  examined, 
the  "  completion  method  "  as  a  rule  leads  to  the  positive  demon- 
stration of  the  amboceptors.  The  "  separation  in  the  cold "  on 
the  contrary,  owing  to  the  peculiarity  of  the  combining  relations 
of  the  separate  components,  is  entirely  inapplicable  in  a  number 
of  cases. 

Gruber,  the  second  author  to  come  out  against  the  conception  of 
the  complex  nature  of  normal  serum  hsemolysins,  sought  to  demon- 
strate amboceptors  in  a  number  of  normal  sera,  by  means  of  "  sepa- 
ration in  the  cold."  In  view  of  the  preceding  it  is  not  surprising  that 
he  failed  in  a  number  of  cases  to  effect  a  separation  of  the  haemolysin. 

Ehrlich  and  Morgenroth  in  their  second  communication  on  hae- 
molysins  have  already  analyzed  the  conditions  for  separating  the 
interbody  by  means  of  absorption,  emphasizing  "  that  the  solution 
of  the  problem  therefore  is  now  possible  only  under  either  of  the  two 
above  mentioned  favorable  conditions;  (1)  When  the  two  haptophore 
groups  of  the  interbody  differ  greatly  in  their  affinity;  and  (2)  when} 
by  means  of  a  combination  whose  discovery  depends  on  chance,  an  acti- 
vating complement  is  found"  • 

The  limitations  of  the  two  methods  applicable  to  an  analysis 
of  the  complex  nature  of  hsemolysins,  are  therefore  sharply  defined. 
In  any  individual  case  when  one  method  fails,  it  will  always  be 
be  necessary  to  make  use  of  the  other  in  order  to  gain  an  insight 
into  the  constitution  of  the  haemolysins  at  all  commensurate  with 
the  means  at  our  disposal.  The  schematic  application  of  only  one 

activated  after  heating  to  55°  C.  can  be  shown  to  exist  in  active  horse  -serum 
(which  does  not  dissolve  guinea-pig  blood)  by  combining  and  completing  it  with, 
guinea-pig  serum. 


CONCERNING   ALEXIN   ACTION.  191 

method  can  lead  to  the  greatest  errors.  In  this  respect  a  comparison 
of  the  results  obtained  by  Buchner  and  Gruber,  is  very  instructive, 
for  among  their  cases  are  two  combinations  which  are  designated 
by  the  one  as  positive,  and  by  the  other  negative. 

The  amboceptor  of  rabbit  serum  for  sheep  blood,  which  Buchner, 
because  of  the  failure  to  reactivate  this  with  sheep  serum,  regarded 
as  absent,  Gruber,  by  means  of  the  cold  separation  method,  could 
demonstrate  as  present;  and  for  ox  serum,  whose  amboceptor  Buchner 
had  already  demonstrated  by  the  activation  with  guinea-pig  serum, 
Gruber,  through  the  failure  of  his  cold  absorption  method,  arrived 
at  the  view  of  a  pure  alexin  action. 

In  Gruber's  negative  cases,  which  embrace  the  following  com- 
binations: I,  rabbit  blood — dog  serum;  II,  rabbit  blood — ox  serum; 
III,  gui:  ea-pig  blood — ox  serum,  IV,  rabbit  blood — guinea-pig  serum; 
I  have  systematically  sought  for  sources  of  fitting  complements 
and  have  found  these  in  abundance.  Naturally  in  view  of  the  experi- 
ences above  mentioned  the  inactivation  of  the  sera  was  effected 
at  the  lowest  temperatures  possible;  thus  dog  serum  and  guinea-pig 
serum  at  50°,  ox  serum  at  52°  C.  In  the  following  sera  (in  part  in 
agreement  with  other  previous  experiences)  I  have  found  comple- 
ments suitable  for  activation: 

I.  For  the  amboceptor  of  dog  serum,  acting  on  rabbit  blood;  in  guinea- 
pig  serum,  ox  serum,  goat  serum,  and  sheep  serum. 

II.  For  the  amboceptor   of   ox  serum,  acting  on  rabbit   blood;  in 
guinea-pig  serum,  rabbit  serum,  and  rat  serum. 

III.  For  the  amboceptor  of  ox  serum,  acting  on  guinea-pig  blood; — 
in  guinea-pig  serum,  human  serum,  rat  serum,  horse  serum,  and  to 
a  slight  extent  also  in  sheep  serum 

Naturally  in  all  the"  experiments,  control  tests  were  made  with 
the  active  serum,  which  served  as  complement.  In  the  cases  desig- 
nated as  positive  completion,  this  serum  by  itself  had  to  exert  no 
haBmolytic  action  or  at  least  to  act  in  a  very  much  smaller  degree. 

Gruber's  fourth  negative  case,  rabbit  blood  and  guinea-pig  serum, 
offered  considerable  difficulties  because  the  combination  is  very 
little  or  not  at  all  effective,  and  it  is  probably  because  of  this  that 
Gruber  speaks  of  "  concentrated  guinea-pig  serum."  Among  a  large 
number  of  guinea-pig  sera  examined  for  this  purpose,  we  found 
only  two  sufficiently  hsemolytically  active.  But  here  also,  through 
the  successful  activation  by  means  of  human  and  ox  sera  (sera,  to 
be  sure,  which  by  themselves  dissolve  rabbit  blood,  but  which  still 


192  COLLECTED  STUDIES  IN  IMMUNITY. 

effect  complete  haemolysis  as  complements  in  amounts  in  which  alone 
they  are  entirely  inert)  we  could  furnish  positive  proof  of  the  presence 
of  amboceptors. 

Buchner  and  Gruber  have  therefore  described  a  total  of  seven 
cases  said  to  show  pure  alexin  action;  and  these  cases  were  held 
by  them  to  be  sufficient  to  decide  in  the  negative  the  entire  question 
of  the  complex  nature  of  the  normal  serum  haemolysins.  Against 
this  we  have  in  all  these  cases  brought  positive  proof  that  the  "  alexin," 
conceived  by  Buchner  to  be  a  simple  unit,  always  produces  its  effects 
through  the  co-action  of  two  components,  the  existence  of  which  is 
demonstrable  in  different  ways.  We  must  therefore  uphold  Ehrlich 
and  Morgenroth's  view,  that  normal  and  artificially  produced  hcemo- 
lysins  exert  their  action  according  to  exactly  the  same  mechanism. 

We  do  not  yet  possess  a  method  generally  applicable  to  demon- 
strate the  complex  nature  of  the  haemolysin,  and  even  a  thorough 
analysis,  therefore,  need  not  necessarily  achieve  the  desired  result  in 
every  case.  The  method  adopted  by  Muller1  for  demonstrating 
the  amboceptors  in  chicken  serum,  which  is  hsemolytic  for  rabbit 
blood,  is  of  interest  in  this  connection.  When  the  usual  methods 
failed  he  found  that  bouillon  injections  caused  an  increase  in  the 
amount  of  complement  in  the  chicken  serum  without  affecting  the 
amount  of  amboceptor.  This  led  him  to  recognize  the  complex 
nature  of  the  hsemolysin,  a  fact  confirmed  by  the  successful  activa- 
tion of  heated  chicken  serum  by  means  of  pigeon  serum.  When 
therefore  in  isolated  cases  the  separation  does  not  succeed  accord- 
ing to  the  methods  heretofore  employed,  such  results,  the  product  of 
incomplete  methods,  most  certainly  do  not  argue  for  a  simple  alexin 
action.  We  hope  that  the  employment  of  the  lowest  possible  tem- 
peratures in  inactivation  will  result  in  increasing  "  completion  " 
possibilities  and  make  the  demonstration  of  the  complex  consti- 
tution of  the  hsemolysins  easier  in  difficult  cases.  At  present  this 
demonstration  has  failed  only  in  the  case  of  eel  serum  (which,  to 
be  sure,  is  very  peculiar  in  its  haemolytic  behavior),  for  thus  far  no 
fitting  complements  have  been  found  for  this  serum.  In  all  other 
cases  of  haemolysis  through  normal  sera,  which  have  been  investi- 
gated for  this  purpose,  according  to  our  experiences  positive  proof 
of  the  presence  of  the  amboceptors  has  been  furnished. 

The  normal  bactericidal  sera  also  owe  their  bactericidal  power 

'P.  Miiller.l.  c. 


CONCERNING  ALEXIN  ACTION.  193 

to  the  co-action  of  two  substances.  Pfeiffer1  furnished  the  first 
observations  which  led  to  this  view  when,  in  1895,  he  succeeded 
in  restoring  the  bactericidal  power  of  inactivated  goat  serum  in  the 
peritoneal  cavity  of  a  guinea-pig.  Moxter2  subsequently  demon- 
strated the  presence  of  normal  bacteriolytic  amboceptors  by  means 
of  reactivating  experiments  in  vitro.  And  according  to  the  numerous 
investigations  of  M.  Neisser  and  Wechsberg  in  this  Institute,  all  of 
the  bacteriolysins  of  normal  sera  which  they  investigated,  are  of 
complex  constitution.  This  is  natural  because  in  the  cell-destroying 
properties  of  normal  serum,  as  in  the  development  and  increase  of  these 
properties  through  immunization  the  mechanism  is  exactly  the  same 
in  principle,  although,  owing  to  the  multiplicity  of  the  reaction  products, 
the  action  of  the  latter  appears  more  complex. 

In  my  investigations  of  the  cytotoxic  properties  of  normal  serum 
I  have  included  the  widely  distributed  spermotoxic  function.  Accord- 
ing to  the  unanimous  opinion  of  all  authorities  the  specific  spermo- 
toxin  produced  by  immunization  consists  of  two  substances.  Thus 
far,  however,  this  has  not  been  demonstrated  for  normal  spermo- 
toxin,  and  MetalnikofT3  has  regarded  the  impossibility  of  reacti- 
vating the  heated  normal  spermotoxic  serum  as  an  important  diag- 
nostic means  to  differentiate  the  latter  from  the  specific  immune 
serum.  In  opposition  to  this,  by  means  of  suitable  mixtures,  I 
was  able,  here  also,  to  convince  myself  of  the  complex  nature  of  the 
normal  spermotoxin.  After  the  spermotoxic  property  of  rabbit 
serum  for  guinea-pig  spermotozoa  had  been  destroyed  by  heating 
to  56°  C.,  I  was  able  to  restore  this  by  the  addition  of  guinea-pig 
or  horse  serum  provided  I  mixed  the  inactive  rabbit  serum  and  guinea- 
pig  serum  in  the  proportion  of  3:1  or  3:2.  In  that  case  the  guinea- 
pig  spermatozoa  were  killed  after  12-15  minutes,  whereas,  hi  the 
control  test  with  inactive  rabbit  serum  or  active  guinea-pig  serum 
alone,  the  spermatozoa  showed  lively  movements  even  after  1^-1  i 
hours.  The  proportion  of  amboceptor  and  complement  employed 
by  me  is  in  direct  contrast  to  that  recommended  for  immune  sera  by 
Metchnikoff  and  his  co-workers.  The  reason  for  this  will  be  under- 

1  R.  Pfeiffer,  Weitere  Mittheilungen  iiber  die  Spezifischen  Antikorper  der 
Cholera,  Zeitschr.  f.  Hygiene,  XX,  1895, 

2  Moxter,  Uber    die    Wirkungsweise  der  bacterienauflosenden    Substanzen 
der  thierischen  Safte,  Centralbl.  f.  Bacteriol.,  XXVI,  1899. 

8  Metalnikoff,  Etudes  sur  la  Spermotoxine,  Annales  de  1'Institut  Pasteur, 
1900. 


194  COLLECTED  STUDIES  IN  IMMUNITY. 

stood  when  the  high  degree  of  amboceptor  concentration  in  immune 
sera  is  considered.  In  my  case  larger  amounts  of  guinea-pig  serum 
must  be  avoided,  because  in  large  doses  the  guinea-pig  serum  by 
itself  finally  exerts  a  toxic  action  on  guinea-pig  spermatozoa.  This 
agrees  with  a  statement  of  London  (1.  c.)  that  most  all  normal  sera 
contain  autospermo toxins. 


SUBSEQUENT  NOTE. — In  the  meantime  the  French  translation  of  a  study  by 
London,  which  had  already  been  published  in  Russian,  has  appeared  (Contribu- 
tion a  I'e'tude  des  spermolysines,  Archives  des  Sciences  Biologiques,  T.  IX,  1902), 
which  shows  that  this  investigator  had  also  already  recognized  the  complex  con- 
stitution of  the  normal  spermotoxin. 

Our  views  concerning  the  complex  nature  of  haemoylsins  have  recently  been 
confirmed  by  Flexner  and  Noguchi  through  the  successful  separation  of  am- 
boceptor and  complement  in  the  cold  (Snake  venom  in  relation  to  haemolysis, 
bacteriolysis,  and  toxicity,  Journal  of  Experimental  Medicine,  vol.  VI,  1902). 


XVIII.    CONCERNING  THE  PLURALITY  OF  COMPLE- 
MENTS OF  THE  SERUM.i 

By  Professor  Dr.  P.  EHRLICH  and  Dr.  H.  SACHS. 

THE  continued  study  of  the  hsemolysins  of  blood  serum  has  not 
only  considerably  extended  our  knowledge  of  the  origin  and  mechan- 
ism of  the  immunity  reaction  directed  against  cells,  but  has  revealed 
to  us  an  unsuspected  complexity  of  cellular  metabolism  to  which 
the  numerous  protective  bodies  circulating  in  the  blood  owe  their 
existence.  It  is  probably  everywhere  conceded  at  the  present  day 
that  the  specific  cytotoxins  produced  through  immunization  consist 
of  two  substances,  amboceptor  and  complement;  and  we  must  regard 
it  as  proven  that  the  cytotoxic  substances  in  normal  serum  are  also 
of  complex  constitution.2  A  simple  alexin  action,  in  Buchner's  sense, 
does  not  exist.  But  even  within  the  limits  of  this  complicated  field, 
Ehrlich  and  Morgenroth  through  their  experimental  work,  have 
come  to  a  further  pluralistic  conception,  so  that  the  closer  analysis 
of  the  factors  making  up  the  cytotoxic  function  of  a  serum  is  enor- 
mously complicated.  Thus  it  has  been  found  in  immunization  with 
cells,  that  not  merely  a  single  kind  of  amboceptor  is  developed  in 
the  blood  serum,  but  that  a  large  number  of  different  types  of  ambo- 
ceptors  appear,  which  vary  both  in  their  cytophile  and  complemento- 
phile  groups.  Furthermore,  a  number  of  facts  and  theoretical  con- 
siderations (discussed  in  detail  in  the  Sixth  Haemolysin  commu- 
nication) could  be  satisfactorily  explained  only  by  the  assumption 
of  a  plurality  of  complements,  and  were  absolutely  irreconcilable 
with  the  Unitarian  assumption  of  only  one  complement  in  each  serum. 

After  all  this  one  might  well  regard  the  pluralistic  conception 
as  well  founded,  and  abandon  all  further  theoretic  argument  along 
this  line.  But  Bordet,3  the  strongest  supporter  of  the  Unitarian 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  Nos.  14  and  15. 

2  See  the  previous  study. 

8  Bordet,  Sur  le  mode  d'action  des  scrums  cytolitiques,  etc.  Annales  de 
1'Instit.  Pasteur,  May,  1901. 

195 


196  COLLECTED  STUDIES  IN  IMMUNITY. 

character,  in  a  recent  work  especially  designed  to  refute  the  plural- 
istic view  of  the  complements,  has  published  a  series  of  experiments, 
which  in  his  opinion  necessarily  point  to  a  simple  alexin.  Bordet's 
argument  is  based  on  the  discovery  of  the  interesting  fact  that  blood 
corpuscles  or  bacteria  treated  with  an  inactive  immune  serum  specific 
for  themselves  were  able  to  deprive  a  normal  active  serum  of  all 
its  complement  activity. 

Bordet  sensitized  blood  corpuscles  with  appropriate  amboceptors, 
and  then  exposed  them  to  the  action  of  a  freshly  drawn  normal  serum. 
If  now  he  waited  for  the  occurrence  of  haemolysis  and  then  added 
sensitized  cells,  bacteria,  or  blood  corpuscles  of  different  species, 
they  remained  totally  unchanged,  although  the  serum  that  had  been 
used  as  complement  was  capable  in  its  original  condition  of  destroy- 
ing these  also.  When  fresh  serum  was  first  brought  into  contact 
with  sensitized  bacteria,  similar  results  were  obtained.  The  blood 
corpuscles  subsequently  added  did  not  then  undergo  haemolysis. 

//  such  an  action  on  one  of  the  sensitive  substrata  has  once  taken 
place,  the  active  sera,  as  a  rule,  are  deprived  of  all  their  complement 
functions,  from  which  Bordet  concludes  that  the  destruction  of  the 
most  varied  elements  by  one  and  the  same  serum  must  be  due  to 
a  single  complement. 

It  must  be  acknowledged  that  these  experiments,  which  we  have 
been  able  to  verify  in  numerous  cases,  at  first  sight  seem  to  sup- 
port Bordet's  view.  If  one  assumes  that  a  certain  serum  A,  which 
is  capable  of  complementing  two  different  bodies  B  and  C,  one  bac- 
tericidal and  the  other  haemolytic,  contains  only  a  single  comple- 
ment, Bordet's  results  would  then  most  readily  be  explained  by 
assuming  that  the  two  immune  bodies  are  identical  in  their  com- 
plementophile  groups.  In  that  case,  of  course,  owing  to  the  previous 
exercise  of  the  one  function,  the  available  complement  will  nave 
been  used  up,  so  that  nothing  is  left  for  the  exercise  of  its  second 
function.  But  a  closer  examination  shows  us  that  this  view  is  an 
artificial  one,  and  does  not  correspond  to  the  facts  observed.  For  if 
it  be  assumed  that  this  particular  serum  A  contains  two  different 
complements,  both  of  which  can  be  absorbed  by  the  amboceptors  B 
and  C,  Bordet's  experiment  will  find  an  entirely  different  explana- 
tion. Now  previous  investigations 1  have  shown  that  the  artifi- 
cially developed  immune  sera  are  not  of  simple  constitution,  but 
contain  a  number  of  different  amboceptors  possessing  different  com- 

1  Ehrlich  and  Morgenroth,  p.  56. 


PLURALITY  OF  COMPLEMENTS  OF  THE  SERUM.  197 

i 

plementophile  groups.  To  one,  therefore,  conversant  with  this  con- 
ception, Bordet's  conclusion  cannot  appear  otherwise  than  forced. 
The  unity  of  the  complement  would  only  then  be  demonstrated 
by  Bordet's  experiment  if  in  the  immune  serum  employed  for  absorp- 
tion but  a  single  complementophile  group  came  into  action,  and 
not  a  plurality  of  groups. 

Despite  these  objections  raised  against  Bordet's  evidence,  and  in 
spite  of  Ehrlich  and  Morgenroth's  previous  positive  demonstration 
of  the  plurality  of  the  complements,  it  seemed  advisable,  owing 
to  the  importance  of  the  question,  to  enter  once  more  upon  a  thorough 
investigation  of  the  subject.  We  at  first  confined  ourselves  to  the 
complements  which  effect  the  hsemolytic  actions,  and  have  been 
able  to  bring  forward  a  large  number  of  new  and  more  conclusive 
proofs  for  the  diversity  of  these  complements  in  the  same  serum. 
These  investigations  have  in  part  already  been  mentioned  by  Ehrlich 
at  the  Congress  of  Naturalists  in  Hamburg. 

The  method  of  the  experiments  was  guided  by  the  following 
considerations.  If  only  a  single  complement  is  present  in  a  cer- 
tain serum,  it  follows  that  all  the  complement  actions  of  this  serum 
would  be  weakened  equally  by  any  given  influence,  chemical,  physi- 
cal, or  thermic.  If,  on  the  contrary,  our  view  of  the  plurality  of 
complements  is  correct,  it  should  be  possible  through  appropriate 
experimental  conditions  to  influence  the  serum  in  such  a  way  that 
only  a  part  of  the  complements  will  be  destroyed,  while  others  remain 
intact.  Not  only  the  absolute  inhibition  of  the  action  of  a  few  com- 
plements, but  also  marked  quantitative  differences  in  the  impair- 
ment of  the  individual  completions  can  only  be  satisfactorily  explained 
by  the  assumption  of  different  substances  as  carriers  of  these  prop- 
erties. A  single  complement  would  have  all  its  functions  impaired 
equally. 

We  have  especially  subjected  the  complementing  property  of 
goat  serum  to  a  thorough  analysis,  using  for  this  purpose  five  different 
combinations  which  can  be  activated  by  goat  serum.  For  simplicity's 
sake,  we  shall  designate  them  by  the  following  numbers : 

Case      I.  Guinea-pig  blood — inactive  normal  goat  serum. 

Case    II.  Rabbit  blood — inactive  normal  goat  serum. 

Case  III.  Rabbit  blood — inactive  serum  of  goats  immunized  with 
rabbit  blood. 

Case  IV.  Ox  blood — inactive  serum  of  goats  immunized  with 
ox  blood. 


198 


COLLECTED  STUDIES  IN  IMMUNITY. 


Case   V.  Dog  blood — inactive   serum  of  goats  immunized  with 

dog  blood. 

The  various  means  by  which  we  have  succeeded  in  a  separation  of 
the  single  complements  are  as  follows: 

1.  Digestion  with  papain. 

2.  Partial  destruction  with  an  alkali. 

3.  Partial  destruction  by  heating  to  50°  C. 

4.  Combination  with  blood-cells. 

We  discovered  that  invariably  under  the  influence  of  papain 
digestion  four  complementing  actions  disappeared,  or  were  more  or 
less  strongly  diminished.  Only  a  single  complement  remained  intact, 
namely,  that  fitting  the  amboceptor  developed  in  goat  serum  through 
immunization  with  rabbit  blood. 

In  these  experiments  20  cc.  goat  serum  mixed  with  3  cc.  of  a  10% 
papain  solution  were  placed  in  an  incubator  in  order  to  digest  the 
complements.  We  found  that  the  proper  time  to  interrupt  the 
digestive  process  was  usually  thirty  to  forty-five  minutes  later, 
when  an  examination1  demonstrated  complete  preservation  of  the 
complements  for  Case  III  with  complete  disappearance  or  consider- 
able diminution  of  the  others.  Of  the  large  number  of  our  experi- 
ments made  in  this  connection  three  examples  may  be  cited.  See 
Table  I. 

TABLE  I. 
DIGESTION  OP  GOAT  SERUM  BY  MEANS  OP  PAPAIN. 


Solvent  Power  of  the  Goat  Serum. 

Example  I. 

Example  II. 

Example  III. 

(a)  Digested. 

(6)  Normal. 

(a)  Digested 

(6)  Normal. 

(a)  Digested. 

(6)  Normal. 

pflQA      T  / 

0.5 

0.25 

0.5 

0.15 

0.5 

0.25 

moderate 

complete 

trace 

complete 

moderate 

complete 

Casp    TT  / 

1.0 

0.5 

1.0 

0.25 

1.0 

0.5 

trace 

complete 

0 

complete 

ft.  trace 

complete 

Case  III 

0.2 
complete 

0.15 
complete 

0.15 
complete 

0.15 
complete 

0.15 
complete 

0.15 
complete 

Case  IV 

0.3 
little 

0.06 
complete 

0.3 
little 

0.07 
complete 

0.5 
strong 

0.08 
complete 

Poop        V 

0.5 

0.06 

0.3 

0.05 

trace 

complete 

alm't  c'm'te 

complete 

1  In  all  our  experiments  the  amount  of  blood  used  as  a  reagent  was  1  cc. 
of  a  5%  suspension. 


PLURALITY  OF  COMPLEMENTS  OF  THE  SERUM. 


199 


When  the  papain  was  allowed  to  act  longer,  the  resistant  com- 
plement III  was  also  affected,  so  that  usually  after  one  and  one-half  to 
two  hours  digestion,  the  goat  serum  was  entirely  deprived  of  all  its 
complements. 

Treatment  with  alkali  in  place  of  papain  digestion  gave  analogous 
results.  We  made  use  of  soda  and  proceeded  as  follows:  10  cc. 
goat  serum  to  w"  ich  1  cc.  7%  soda  solution  had  been  added,  were 
kept  in  the  incubator  for  one  and  one-quarter  hours,  and  then  neutral- 
ized with  hydrochloric  acid.  The  solvent  power  was  compared  with 
a  goat  serum  which  by  the  simultaneous  addition  of  soda  and  hydro- 
chloric acid,  had  been  brought  to  the  same  concentration  of  salt 
without  having  been  subjected  to  the  damaging  influence  of  the 
soda.1  (See  Table  II.) 

TABLE  II. 
DESTRUCTION  OF  THE  GOAT  SERUM  BY  MEAXS  OP  SODA. 


Solvent  Power  of  the 

Goat  Serum. 

(a) 

(6) 
Normally. 

After  Soda  Treatment. 

Case 

I 

0 

.5 

0 

0 

.1 

complete 

tt 

11 

1 

.0 

0 

0 

.6 

1 

n 

III 

0 

.12 

complete 

0 

.03 

• 

' 

tt 

IV 

0 

.5 

0 

0 

.04 

• 

' 

V 

0 

.3 

0 

0 

.04 

Hence,  owing  to  the  action  of  the  soda  the  complements  for  Cases  I, 
II,  IV,  and  V  have  completely  disappeared,  whereas  Complement  III 
is  still  present,  although  its  action  is  but  one-fourth  of  what  it  was. 

We  have  furthermore  effected  a  separation  of  the  complements  by 
heating  the  goat  serum  to  49°-50°  C.  for  half  an  hour.  At  this  tempera- 
ture the  solvent  action  of  normal  goat  serum  for  rabbit  and  guinea- 
pig  blood  has  been  completely  destroyed  or  almost  so.  On  the  other 
hand,  the  complement  action  for  the  artificially  produced  immune 
bodies  is  more  or  less  preserved,  as  can  be  seen  from  Table  III. 

The  experiment  shows  that  in  this  case  complement  IV  is  the 
most  resistant,  in  contrast  to  its  behavior  with  papain  or  soda.  In 
the  two  latter  cases,  complement  III  had  shown  itself  the  most  resist- 
ant. If  we  examine  the  table  more  closely  we  shall  further  see  a 

1  The  resulting  salt  concentration,  by  the  way,  is  so  slight  that  the  solvent 
power  was  in  no  way  decreased  thereby. 


200 


COLLECTED  STUDIES   IN  IMMUNITY. 


TABLE  III. 
HALF  AN  HOUR'S  HEATING  OF  THE  GOAT  SERUM  TO  50°  C. 


Solvent  Power  of  the  Goat  Serum. 

What  is  Left  of  the 
Original  Solvent 
Power. 

(a) 
Heated. 

(6) 
Normally. 

Case     I 

"       II 
"     III 
"     IV 
V 

1.0  trace 
1.0     " 
0.08  complete 
0.035 
0.75 

0  .  1     complete 
0  .  25 
0.01 
0.035       " 
0.02 

>    almost  nothing 

1 

* 

difference  in  the  diminution  suffered  by  complement  V  and  that 
suffered  by  complement  III.  This  is  so  marked  that  merely  a  com- 
bination of  the  above  three  experiments  already  furnishes  positive 
proof  that  the  complement  actions  in  III,  IV,  and  V  proceed  inde- 
pendently of  one  another,  and  are  effected  by  three  different  comple- 
ments. 

But  against  this  method  of  proof  the  objection  might  be  made 
that  in  the  end  we  may  still  be  dealing  with  simple  [einheitlich]  com- 
plements and  that  the  results  of  the  experiments  mentioned  do  not 
necessarily  indicate  a  plurality  of  complements.  It  could  be  assumed 
that  the  view  we  have  expressed  concerning  the  plurality  of  the 
complements  was  true  only  in  a  certain  restricted  sense.  Thus  it 
would  be  possible  that  the  complements  possessed  but  one  hapto- 
phore  group,  but  a  plurality  of  zymotoxic  groups  of  which  one  effected 
the  damaging  action  in  any  individual  case.  It  could  then  easily 
be  imagined  that  the  various  zymotoxic  groups  differ  from  one  another 
in  their  behavior  toward  chemic  or  thermic  influences,  so  that  per- 
haps one  was  injured  by  papain,  and  another  by  an  alkali.  In  order 
to  decide  this  possibility  either  one  way  or  another  it  seemed  advis- 
able to  undertake  absorption  experiments.  In  case  of  a  simple 
complement  with  different  zymotoxic  groups,  the  complement  would 
be  absorbed  as  a  unit,  whereas  in  the  other  case,  differences  such 
as  we  have  already  observed  on  heating,  etc.,  would  be  expected  to 
occur. 

Because  of  the  great  significance  of  obsorption,  we  regard  these 
experiments  as  particularly  valuable.  Our  first  experiments  were 
made  to  see  if  the  complements,  like  so  many  bodies  known  to  chem- 
istry would  adhere  to  granular  substances  of  various  kinds  by  virtue 
of  surface  attraction.^  Bone  charcoal,  skin  powder,  lycopodium, 


PLURALITY  OF  COMPLEMENTS  OF  THE  SERUM.  201 

and  diatom  earth,  which  we  employed  for  this  purpose,  all  proved 
more  or  less  unsuitable  for  the  absorption  of  complement.  A  stronger 
absorbent  power  on  the  other  hand  was  exhibited  by  organized  mate- 
rials, thus  confirming  the  statements  of  von  Dungern.1  Suspensions 
of  staphylococci,  when  used  in  sufficient  quantity,  were  able  to  abstract 
the  complements  quite  energetically.2  In  like  manner  yeast  powder 
is  an  excellent  means  to  deprive  a  serum  of  its  complement  prop- 
erties. A  separation  of  the  complements,  however,  was  not  achieved 
by  these  experiments. 

We  are  inclined  to  believe  that  in  these  cases  the  fixation  of  the 
complements  is  due  to  physical  absorption  and  not  to  definite  chemi- 
cal union.  This  view  is  the  outcome  of  the  positive  results  obtained 
in  the  absorptions  when  we  employed  blood-cells  which  had  been, 
mixed  with  suitable  amboceptors,  and  which,  according  to  our  views,, 
were  able  to  bind  complements  chemically.  If  blood-cells  which 
have  been  saturated  (sensitized)  with  a  normal  immune  body  or 
with  one  artificially  produced  are  shaken  with  a  certain  amount3 
of  complementing  serum,  it  is  very  easy  to  determine  that  in  con- 
formity with  the  results  of  Bordet's  experiments,  the  complement 
properties  possessed  by  the  normal  serum  have  in  most  cases  com- 
pletely disappeared  with  the  onset  of  haBmolysis.  It  was  just  this 
phenomenon  that  led  Bordet  to  his  Unitarian  conception.  Yet  even 
in  this  absorption  it  is  possible  by  means  of  suitable  methods  to 
convince  one's  self  of  the  diversity  of  the  complements,  for  by  making 
the  time  as  short  as  possible  only  those  complements  are  absorbed 
which  possess  the  strongest  affinity  for  corresponding  complementophile 
groups.  Naturally  experiments  of  this  kind  are  difficult  and  require 
considerable  variation.  But  it  is  usually  possible  to  finally  devise 
a  suitable  method  of  procedure.  An  interesting  case  studied  by 
us  in  this  respect  is  the  combination  rabbit  blood  and  goat  serum 
(Case  II).  With  sufficiently  rapid  digestion  (2  to  3  minutes  at  the 
most,  possibly  with  the  aid  of  gentle  heat)  the  decanted  portion 
showed  a  considerable  loss  of  complements  for  Case  IV  or  V,  or  for 
both,  without  suffering  any  injury  in  the  rest  of  its  complement 

1  See  p.  36. 

2  The  same  results  were  obtained   by  Wilde   (Berl.  klin,  Wochenschr.  1901, 
No.  34)  in  absorption  tests  with  anthrax,  cholera,  and  typhoid   bacteria;    but 
to  conclude  from  this  that  the  alexin  is  a  simple  unit,  as  Wilde  does,  is  not  per- 
missible in  view  of  our  above  statements. 

3  This  amount  must  be  determined  separately  for  each  case. 


202 


COLLECTED  STUDIES  IN  IMMUNITY. 


functions.    We  were  able  to  observe  this  behavior  repeatedly  and 
reproduce  the  following  as  an  illustration. 

10  cc.  goat  serum  are  shaken  with  8  cc.  rabbit  blood  for  a  very 
short  time  and  then  rapidly  centrifuged.  The  following  table  shows 
the  solvent  power  of  the  decanted  fluid  and  of  normal  goat  serum. 
The  figures,  I-V,  correspond  to  the  blood-cell  amboceptor  com- 
bination employed  in  the  previous  tables. 

TABLE  IV. 
BRIEF  ABSORPTION  OP  GOAT  SERUM  WITH  RABBIT  BLOOD. 


Solvent  Power  of  the  Goat  Serum. 

(a) 
After  the  Absorption. 

(6) 
Normally. 

Case      I 
"      II 
"     III 
"     IV 

11       V 

0  .  25  complete 
0.5 
0.04 
0.35  complete 
0.2 

0  .  25  complete 
0.5 
0.04        " 
0.08  complete 
0.03 

Complements  I,  II,  and  III  have  been  completely  preserved, 
IV  and  V  have  been  reduced  to  one-fourth  and  one-seventh  respec- 
tively, thus  furnishing  another  proof  for  their  diversity.  It  is  of 
special  interest  that  in  this  brief  action  the  particular  activating 
principle  (complement  II)  which  we  shall  term  the  "  dominant  com- 
plement "  has  not  at  all  combined  with  the  cell,  whereas  other  com- 
plements, which  are  of  no  consequence  so  far  as  the  solvent  process 
is  concerned,  have  already  been  subjected  to  a  distinct  absorption. 

With  the  absorptions  are  also  to  be  classed  the  experiments  con- 
cerning Case  I,  which  we  have  made  with  guinea-pig  blood  stro- 
mata  obtained  after  the  method  of  H.  Sachs  l  by  heating  the  blood 
to  55°  C.  In  these  stromata  the  receptors  which  bind  the  ambo- 
ceptors  present  in  normal  goat  serum  have  been  preserved  capable 
of  reacting. 

These  experiments  demonstrated  the  absorption  of  the  comple- 
ments for  the  two  normal  hsemolysins  (Cases  I  and  II)  while  the 
rest  of  the  complements  were  in  the  main  preserved.2  An  experi- 
ment of  this  kind  is  shown  in  Table  V. 

1  See  page  167. 

2  In  this  also  it  is  necessary  first  to  determine  the  favorable  conditions 
governing  the  experiment.     Thus,  in  order  to  completely  bind  the  guinea-pig 


PLURALITY  OF  COMPLEMENTS  OF  THE  SERUM. 


203 


20  cc.  goat  blood  are  treated  with  the  stromata  from  53  cc.  guinea- 
pig  blood.  After  absorption  has  occurred  the  mixture  is  centrifuged 
and  the  complement  action  of  the  fluid  compared  with  that  of  nor- 
mal goat  serum.  (See  Table  V.) 

TABLE  V. 
ABSORPTION  OP  THE  GOAT  SERUM  BY  GUINEA-PIG  BLOOD  STROMATA. 


Solvent    Power 

(a)  Of  the  Decanted 
Fluid. 

(6)  Of  the  Normal 
Goat  Serum. 

Case     I 
"      II 
"     III 
"     IV 
"       V 

1  .  0  faint  trace 
1.0     " 
0.1  complete 
0.15  complete 
0.15  complete 

0.15  complete 
0.25        " 
0.1  complete 
0.04  complete 
0.15  complete 

Hence  after  the  absorption,  the  complements  of  the  normal  haemo- 
lysins  had  almost  completely  disappeared,  while  complements  III 
and  V  were  entirely  preserved.  Complement  IV  occupies  a  place 
between  these,  for  in  this  case  also  a  partial  absorption  could  not 
be  avoided.  Its  behavior  very  prettily  confirms  the  demonstra- 
tional  ready  made  by  us  of  this  complement's  peculiar  isolated 
position. 

Entirely  analogous  results  are  obtained  when,  instead  of  using 
-guinea-pig  blood  stromata,  a  series  of  experiments  is  made  with 
red  blood-cells,  using  the  red  fluid  obtained  when  the  red  blood-cells 
have  dissolved  directly  as  complement  for  another  combination. 
In  such  experiments  we  could  show  that  the  blood  solution  thus 
obtained  had  lost  complements  I  and  II  and  contained  only  the 
complements  for  cases  III,  IV  and  V.  This  method  of  procedure 


blood  hsemolysin  (amboceptor+  complement)  of  normal  goat-blood  serum, 
it  is  necessary  to  absorb  with  a  large  excess  of  guinea-pig  blood  stromata.  It 
then  readily  happens  that  some  complements  other  than  those  belonging  to 
the  two  normal  hsemolysins  suffer  injury  to  a  greater  or  less  extent.  This 
was  observed  especially  in  several  cases  in  which,  in  order  to  render  easier  the 
complete  binding  of  the  complements  for  the  normal  hsemolysins,  the  guinea- 
pig  blood  stromata  had  been  sensitized  with  a  large  amount  of  inactivated 
normal  goat  serum.  In  that  case,  evidently,  several  partial  amboceptors  present 
in  the  goat  serum  in  relatively  small  amounts  and  possessing  affinities  also 
for  the  other  complements  come  into  play. 


204 


COLLECTED  STUDIES  IN  IMMUNITY. 


therefore  confirms  the  separation  effected  by  means  of  the  stromata,. 
whereby  the  complements  of  the  normal  hsemolysins  I  and  II  are 
separated  from  the  rest. 

Bordet  himself,  by  the  way,  has  described  such  a  case  concerning 
the  combination  rabbit  blood — guinea-pig  serum.  This  experiment, 
of  course,  was  not  to  be  reconciled  with  his  Unitarian  view,  and  he 
therefore  sought  to  explain  this  inconvenient  result  in  accordance 
with  his  view  by  assuming  a  special  law  of  distribution  for  the 
normal  ha3molysins,  together  possibly  with  an  inhibiting  action 
exerted  by  the  products  of  the  destruction  of  the  red  blood-cells 
first  used,  on  further  solution  of  the  same.1  Against  this  we  should 
like  to  emphasize  that  in  our  case  the  result  has  been  confirmed  by 
the  experiment  with  blood  stromata.  By  means  of  this,  since  the 
stromata  plus  the  anchored  complement  is  removed  by  centrifuging, 
Bordet 's  assumptions  can  be  entirely  excluded. 

Our  absorption  experiments  therefore  show  that  of  the  two  possi- 
bilities, namely,  of  a  complement  with  several  different  zymotoxic  groups, 
or  of  a  plurality  of  different  complements,  the  latter  assumption  must 
be  accepted. 

Regarding  the  number  of  complements  to  be  assumed  for  normal 
goat  serum,  as  based  on  our  experiments,  this  can  best  be  seen  from 
the  following  table: 


TABLE  VI. 


Complementing  Power   of   Goat   Serum   after 

(a) 

(&) 

(c) 

(d) 

(e) 

(/) 

Digestion 
with 
Papain. 

The    Action 
of  Soda. 

Heating 
to  500. 

Absorption 
with 
Rabbit 
Blood. 

Absorption 
with 
Guinea-pig 
Blood: 

Absorption 
with 
Guinea-pig 
Blood 

Stromata. 

Case     I 

0 

0 

0 

+ 

0 

0 

"      II 

0 

0 

0 

-j. 

0 

0 

"     III 

_j_ 

_j_ 

i 

i 

"     IV 

0 

0 

-f 

1 

_l_ 

1 

"       V 

0 

0 

* 

1 

+ 

+ 

1  This  objection,  moreover,  is  entirely  incomprehensible  to  us.  According 
to  our  view,  normal  and  artificially  produced  hsemolysins  manifest  their  action 
by  means  of  the  same  mechanism;  for  when  the  normal  amboceptors  are  re- 
placed by  the  host  of  amboceptors  present  in  an  immune  serum,  new  comple- 
mentophile  groups  come  into  action,  and  with  these,  of  course,  new  partial 
complements. 


PLURALITY   OF  COMPLEMENTS  OF  THE  SERUM.  205 

This  shows  us  that  the  two  complements  I  and  II  (normal  haemoly- 
sins)  cannot  by  these  experiments  be  differentiated  from  each  other, 
that  the  other  three  complements,  however,  can  absolutely  be  distinguished 
by  their  behavior,  not  only  from  one  another  but  also  from  the  first  group. 
Hence  in  the  five  different  combinations  the  existence  of  at  least  four 
different  complements  is  positively  demonstrated.  And  that  the  two 
normal  hsemolytic  functions  of  goat  serum  are  also  effected  by  two 
different  complements  follows  from  a  previous  experiment  of  Erhlich 
and  Morgenroth.1  These  authors  showed  by  filtering  a  normal  goat 
serum  through  Pukall  filters,  that  the  filtrate  contained  exactly  the 
same  amount  of  complement  for  guinea-pig  blood,  whereas  the  com- 
plement for  rabbit  blood  was  almost  entirely  absent.  E.  Neisser 
and  Doring  2  have  confirmed  this  result  in  the  case  of  human  serum. 

The  necessary  consequence,  therefore,  of  our  experiences  with 
goat  serum  is  the  demonstration  of  the  fact  that  in  the  five  completions 
examined,  five  different  complements  of  the  goat  serum  come  into  play.3 

We  have  also  examined  the  complementing  properties  of  the 
sera  of  other  animal  species,  and  have  arrived  at  results  which  abso- 
lutely contradict  the  Unitarian  view  of  the  complements.  These 
experiments  concern  first  the  serum  of  rabbits.  We  shall  proceed 
from  the  fact  determined  by  Schiitze  and  Scheller4  under  Wasser- 
mann's  direction,  that,  following  intravenous  injections  of  goat  blood, 
the  rabbit  serum  completely  loses  its  property  to  dissolve  goat  blood. 

The  question  now  was  whether  the  rabbit  serum  had  been 
deprived  merely  of  this  one  complementing  function,  or  whether  it 
had  also  suffered  loss  in  the  rest  of  its  complement  properties. 

We  therefore  tested  the  power  of  rabbit  serum,  before  and  after 
the  injection  of  goat  blood,  to  activate  the  immune  body  obtained 
by  immunizing  rabbits  with  ox  blood.  As  the  essential  result  of 
our  numerous  investigations  we  established  the  fact  that  the  com- 

1  See  page  56. 

2  E.  Neisser  and  Doring,  Berl.  klin.  Wochenschr.  1901,  No.  22. 

3  Through  the   courtesy  of   Dr.  Wendelstadt  in  Bonn,  we  learn  that  that 
investigator,  by  means  of  an  interesting  method,  has  also  succeeded  in  demon- 
strating a  number  of  complements  in  goat  serum.     He  immunized  a  goat  with 
several  species  of  blood  and  was  then  able  by  means  of  chemical  and  thermic 
influences  to  separate  the  complements  fitting  the  immune  bodies  produced. 
See  Centralblatt  f.  Bacteriologie,  in  which  this  study  is  about  to  appear. 

4  Schutze     and     Scheller,   Experimentelle    Beitrage    zur    Kenntniss  der  im 
normalen  serum  vorkommenden  globuliciden  Substanzen,  Zeitschrift  f.  Hygiene, 
Vol.  36,  1901. 


2H6 


COLLECTED   STUDIES  IN  IMMUNITY. 


plement  for  goat  blood  disappeared  after  the  injection  while  that 
for  the  immune  body  sensitizing  ox  blood  remained  intact.  The 
following  test  may  serve  as  an  example: 

A  rabbit  of  1900  g.  is  injected  intravenously  with  22  cc.  goat 
blood.  The  change  in  the  solvent  power  of  the  goat  serum  which 
results  from  the  injection  may  be  seen  from  the  following  table: 

TABLE  VII. 


Solvent  Power  of 

the  Rabbit  Serum. 

Blood  Species. 

(a) 
Before  the  Injection. 

(&) 
After  the  Injection. 

Goat  blood  —  inactive  normal  rabbit  serum 
Ox  blood  —  inactive  serum  of  a  rabbit  im- 
munized with  ox  blood 

0.35  complete 
0  05        " 

1  .  0  no  solution 
0  25  complete 

Similar  results  are  obtained  in  the  absorption  of  rabbit  serum 
by  means  of  goat  blood  in  vitro,  so  that  this  experiment  already  justi- 
fies us  in  assuming  two  different  complements  in  rabbit  serum. 

In  one  of  these  experiments  with  goat-blood  injections  the  hae- 
molysis of  pig  blood  by  means  of  rabbit  serum  was  also  tested,  and 
it  was  found  that  the  complement  of  the  normal  hsemolysin  for  pig 
blood,  like  that  for  sensitized  ox  blood,  had  remained  unchanged. 
Neither  was  it  possible  by  means  of  intravenous  injection  of  pig 
blood  to  separate  these  two  complements  of  rabbit  serum,  for  in 
this  case,  contrary  to  their  previous  behavior,  both  were  absorbed, 
while  the  complement  for  goat  blood  remained  in  the  serum.  For 
the  present  we  must  therefore  content  ourselves  with  the  knowledge 
that  we  have  brought  forward  positive  proof  of  the  existence  of  two- 
different  complements  in  rabbit  serum;  a  proof  which  is  strongly  cor- 
roborated by  the  divergent  behavior  of  the  two  complements  in  the 
absorption  with  goat  blood  and  pig  blood  respectively. 

The  difference  between  the  two  complements  also  manifests 
itself  in  their  different  vulnerability  to  papain.  While  the  com- 
plementing power  of  rabbit  serum  toward  the  artificially  produced 
immune  body  for  ox  blood  suffers  considerable  diminution  under  the 
influence  of  papain  digestion,  the  complement  of  normal  hsemolysin 
for  goat  blood  is  hardly  affected,  so  that  this  experiment  also  sub- 
stantiates our  demonstration  of  at  least  two  complements  in  rabbit 
serum. 

Some  rather  cursory  tests  were  finally  made  with  dog  and  guinea- 


PLURALITY  OF  COMPLEMENTS  OF  THE  SERUM. 


207 


pig  serum  with  the  view  of  separating  the  complements  by  care- 
fully heating  the  sera.  In  the  dog  serum  a  half  hour's  heating  to 
49.5°  and  in  the  guinea-pig  serum  to  49°  was  sufficient  to  enable 
us,  by  means  of  the  differences  of  the  weakening  of  the  various  com- 
plementing functions,  to  recognize  here  also  the  plurality  of  the  com- 
plements. The  results  of  these  experiments  are  shown  in  Tables  VIII 

and  IX. 

TABLE  VIII. 
HALF  AN  HOUR'S  HEATING  OF  DOG  SERUM  TO  49°.5  C. 


Solvent  Power  of 

the  Dog  Serum. 

Solvent  Power 

(a)  Heated. 

(6)  Normal. 

Still  Preserved. 

I.  Rabbit  blood  —  inactive  dog 
serum  

05         0 

0  .  25  complete 

o 

II.  Guinea-pig  blood  —  inactive 
dog  serum  .... 

05       0 

01        " 

o 

III.  Sheep  blood  —  inactive  dog 
serum  

05       0 

0  08      " 

o 

IV.  Human  blood  —  inactive  se- 
rum of  goats  immunized 
with  human  blood  

0  5  moderate 

0  15      " 

less  than  £ 

V  Ox  blood  —  inactive  serum  of 
goats  immunized  with  ox 
blood  

0  35  complete 

0  06      " 

4 

VI.  Ox  blood  —  inactive  serum  of 
rabbits  immunized  with  ox 
blood 

0  5  strong 

0  045    " 

less  than  -fa 

TABLE  IX. 
HALF  AN  HOUR'S  HEATING  OF  THE  GUINEA-PIG  SERUM  TO  49°  C. 


Blood-cell  —  Amboceptor  Combination. 

Solvent  Power  of  the  Guinea-pig 
Serum. 

Solvent  Power 
Still  Preserved. 

(a)  Heated  to  49°. 

(6)  Normal. 

I.  Rabbit       blood  —  inactive 
guinea-pig  serum  
II.  Ox  blood  —  inactive  guinea- 
pig  serum 

1.0               0 

0  .  5        trace 
0.008  complete 
0.025        " 
0.025        " 
0.5 

0  .  5    complete 
0.5 

0.008      " 
0.025      " 
0.006      " 
0.25 

0 

almost  0 

1 

1 

i 
Q 

i 

III.  Ox  blood  —  inactive  serum  of 
goats  immunized  with  ox 
blood  

IV.  Ox   blood  —  inactive    serum 
of  rabbits  immunized  with 
ox  blood  

V.  Sheep    blood  —  inactive    se- 
rum of  goats  immunized 
with  sheep  blood  
VI.  Dog  blood  —  inactive  serum 
of  goats  immunized  with 
dog  blood  

208  COLLECTED  STUDIES  IN  IMMUNITY. 

If  we  review  all  our  observations,  they  show  that  in  the  ques- 
tion of  the  complements  the  Unitarian  conception  leads  to  a  con- 
fused mass  of  inexplicable  contradictions,  and  that  it  must  there- 
fore be  abandoned.  All  experiences,  on  the  other  hand,  harmonize 
best  with  the  assumption  of  a  number  of  different  complements  in  the 
.same  serum.  Sober  consideration,  in  fact,  makes  this  appear  as 
the  necessary  consequence  of  such  a  multiplicity  as  has  been  demon- 
strated anew  by  these  experiments.  It  is  a  satisfaction  to  know 
that  in  the  Institut  Pasteur  a  high  authority  (Metchnikoff )  *  has 
also  given  up  the  Buchner-Bordet  conception  of  the  simplicity 
[einheitlichkeit]  of  the  alexines,  and  has  come  to  the  conclusion 
that  there  are  at  least  two  complements  in  the  same  serum.  Metch- 
nikoff found  that  the  exudates  rich  in  macrophages  acted  hsemo- 
lytically,  but  were  unable  to  effect  bacteriolysis.  On  the  other  hand 
the  exudates  rich  in  microphages  exerted  a  marked  bactericidal  action, 
but  were  incapable  of  dissolving  even  sensitized  red  blood-cells. 
Metchnikoff  concludes  that  these  two  kinds  of  cells  produce  two 
different  complements,  one,  which  he  terms  microcytase,  effects  the 
bacteriolytic  actions,  the  other,  macrocytase,  is  the  carrier  of  the 
functions  which  destroy  animal  cells.  He  emphasizes  that  the 
demonstration  of  the  duality  of  complements  does  not  affect  the 
correctness  of  Bordet's  experiments,  and  he  says  in  explanation  of 
Bordet's  results:  "  II  n'y  a  qu'a  admettre  que  les  elements  figures, 
une  fois  qu'ils  sont  impregne's  de  fixateurs  specifiques,  deviennent 
capables  d'absorber  non  seulement  la  cytase  qui  les  digere,  mais 
aussi  une  autre  qui,  sans  les  dissoudre,  se  fixe  simplement  sur  eux." 

So  far  as  this  is  concerned  we  should  like  again  to  emphasize 
that  we  also  have  not  questioned  the  correctness  of  Bordet's  experi- 
ments, but  have  merely  objected  to  the  Unitarian  conception  deduced 
therefrom.  The  old  controversy  concerning  the  two  views  would  thus 
<be  ended,  and  definitely  decided  in  favor  of  our  view. 

1  Metchnikoff,  L'Immunite  dans  les  maladies  infectieuses,  page  206,  Paris, 
1901. 


XIX.    CONCERNING  THE  MECHANISM  OF  THE  ACTION 
OF  AMBOCEPTORS.1 

By  Prof.  Dr.  P.  EHRLICH  and  Dr.  H.  SACHS. 

I.  Blocking  of  the  Amboceptor  by  Complementoids. 

THE  complements  which  activate  the  amboceptors  of  blood  serum 
are,  as  is  well  known  from  the  experiments  of  Ehrlich  and  Morgen- 
roth,  like  the  toxins  characterized  by  two  groups  in  the  molecule, 
viz.,  the  haptophore  group,  which  combines  with  the  complemento- 
phile  group  of  the  amboceptor,  and  the  zymotoxic  group,  which 
represents  the  actual  carrier  of  the  complement's  specific  function. 
In  complete  harmony  with  this,  Ehrlich  and  Morgenroth2  could 
show  through  the  production  of  anticomplements  by  heating  inac- 
tivated sera,  that  the  complements,  like  the  toxins,  under  certain 
circumstances  are  changed  into  inactive  modifications.  These  mod- 
ifications are  still  able  to  excite  the  production  of  antibodies,  and 
must  therefore  possess  their  haptophore  group  intact;  in  analogy 
with  the  toxoids,  therefore,  they  are  called  complementoids.  Although 
the  presence  of  the  complementoids  could  easily  be  shown  by  means 
of  animal  experiments,  it  was  impossible  to  demonstrate  their  react- 
ing power  by  means  of  haemolytic  test-tube  experiments.  The 
reason  for  this  was  that  a  decrease  of  the  complement  action,  such 
as  was  to  be  expected  in  the  inactivated  sera  (which  really  con- 
stitute a  mixture  of  amboceptor  and  complementoid),  did  not  occur, 
even  when  the  complementoid  was  present  in  large  amounts.  Ehr- 
lich and  Morgenroth  have  therefore  assumed  that  in  the  change  from 
complement  to  complementoid,  the  affinity  of  the  complement's  hap- 
tophore group  suffers  a  diminution.  A  similar  assumption  has  been 
made  by  Myers3  for  the  toxoids  of  cobra  poison. 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  No.  21. 

2  See  page  79. 

8  Myers,  Cobra  Poisons,  etc.,  The  Lancet,  1898. 

209 


210  COLLECTED  STUDIES  IN  IMMUNITY. 

It  is,  of  course,  not  at  all  necessary  that  such  a  diminution  of 
affinity  occur  with  all  complements;  and,  considering  the  great  dis- 
tribution and  multiplicity  of  the  substances  included  in  the  con- 
cept "  complement,"  this  is  a  priori  but  little  probable.  We  have 
therefore  hoped  that  in  the  course  of  our  investigations  we  would 
discover  a  suitable  combination  in  which,  on  the  formation  of  com- 
plementoid,  the  diminution  of  affinity  does  not  occur,  or  occurs  only 
to  a  slight  degree.  As  a  matter  of  fact,  such  a  case  has  recently 
presented  itself  to  us. 

As  is  well  known,  normal  dog  serum  dissolves  guinea-pig  blood 
energetically.  If  this  dog  serum  is  inactivated,  it  is  easy  to  restore 
the  h£emolytic  property  with  active  guinea-pig  serum;  the  inacti- 
vation,  however,  must  be  effected  at  suitable  temperatures,  50-51°  C., 
for  at  higher  temperatures,  as  Sachs  1  has  demonstrated,  the  ambo- 
ceptor of  dog  serum  shows  itself  thermolabile. 

That  is  why  Buchner  in  his  experiments  could  not  activate  the 
amboceptor  of  dogs,  for  at  the  inactivating  temperature  employed 
by  him,  60°  C.,  the  completion  with  guinea-pig  serum  is  no  longer 
possible.     Continuing  the  analysis  of  this  interesting  case  we  made 
a  curious  observation:    If  guinea-pig  blood-cells  were  treated  with 
appropriate  amounts  of  inactive  dog  serum  for  one  hour  in  an  incu- 
bator and  the   mixture   then   centrifuged,   it  was  found  that,  con- 
trary to  all  expectations,  the  sediments  could  no  longer  be  activated 
with  guinea-pig  serum,  whereas  when  the  three  constituents  were 
mixed  simultaneously,  prompt  haemolysis  occurred.     (See  Table  I.) 
Our  first  thought  was  that  the  amboceptor,  despite  the  relatively 
long  contact  with  the  blood-cells  (one  hour),  had  perhaps  not  been 
bound  by  these.     Such  behavior,  to  be  sure,  although  conceivable 
and,  as  we  shall  see  later,  sometimes  actually  occurring,  would  be 
exceptional.     In  this  case,  however,  we  could  readily  convince  our- 
selves that  this  suspicion  was  groundless.     For  when  by  means  of 
guinea-pig  serum  we  attempted  to  activate  the  guinea-pig  blood- 
cells  digested  with  dog  serum  as  above  described,  without  first  removing 
the  fluid  medium,  no  haemolysis  took  place.     And  we  could  see  by  the 
behavior  of  the  fluid  obtained  by  centrifuging  the  blood  mixture  as 
described  that  the  amboceptor  was  not  present  in  the  fluid.     When 
this  was  allowed  to  act  on  native  guinea-pig  blood  to  which  active 
guinea-pig  serum  (complement)  had  been  added,  no  solution  could 

1  See  pages  181  et  seq. 


THE  MECHANISM  OF  THE  ACTION  OF  AMBOCEPTORS.     211 
TABLE   I. 


Inactive  Dog 
Serum. 

cc. 

Solvent  Action  on  the  Guinea-pig  Blood.1 

(A) 
Blood  +  Inactive 
Dog  Serum  kept  at 
37°  for  One  Hour, 
then  Centrifuged. 
To  the  Sediments 
0.5  cc. 
Guinea-pig  Serum. 

(B) 

Blood  +  Inactive 
Dog  Serum  +0.5  cc. 
Guinea-pig  Serum 
Mixed 
Simultaneously. 

1.         1.0 
2.         0.5 
3.       0.35 
4.       0.25 
5.       0.15 
6.       0 

0 

complete 

«  « 
<  < 

almost  complete 
0 

*  The  amount  of  blood  used  in  our  experiments  is  always  1  cc.  of  a  5%  suspension  in  .85% 
salt  solution. 

be  effected.     Hence  the  amboceptor  must  have  been  bound  by  the  blood- 
cells. 

How  then,  through  this  previous  binding,  had  the  amboceptor 
lost  its  power  of  being  activated?  After  excluding  all  other  possible 
explanations  we  were  forced  to  conclude  that  the  phenomenon  observed 
is  due  to  a  blocking  of  the  complementophile  groups  of  the  dog  serum's 
amboceptor  by  the  complementoids  still  present  in  the  inactive  serum. 
The  correctness  of  this  view  has  to  our  minds  been  confirmed: 

1.  By  the  isolated  binding  of  the  amboceptor  at  0°  C. 

2.  By  the  subsequent  blocking  of  the  amboceptor  bound  at  0°  C., 
by  means  of  free  complementoids. 

3.  By  the  behavior  of  dog  serum  inactivated  by  shaking  with 
yeast. 

4.  By  the  combining  experiment  with  inactive  dog  serum  (inac- 
tivated by  heat)  when  the  salt  content  of  the  fluids  .was  increased. 

We  shall  take  these  up  in  order. 

1.  If  we  repeated  the  combining  experiment  above  mentioned, 
modifying  it,  however,  so  that  the  amboceptor  was  anchored  by  the 
blood-cells,  not  at  37°  C.,  but  at  0°  C.,  we  could  show  that  the  guinea- 
pig  blood-cells,  treated  in  this  way  at  0°  C.,  were  all  activated  by  guinea- 
pig  serum.  (See  Table  II.) 

Now  we  know  that  at  0°,  as  a  rule,  only  the  amboceptor  is  bound 
by  the  blood-cells,  and  that  the  complement  for  the  most  part  is 
uninfluenced.  It  is,  therefore,  perhaps  quite  natural  in  those  cases 
in  which  the  complementoids,  like  the  complements,  are  bound  by 


212 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE  II. 
GUINEA-PIG   BLOOD. 


Inactive  Dog 
Serum. 

cc. 

Amount   of  Solution  of  the  Sediments 
on  the  Addition  of  0.4  cc.   Guinea- 
pig  Serum,  after  Previously  having 
been  Treated. 

(A)  At  0°. 

(B)  At  37°. 

1.        1.0 
2.       0.5 
3.       0.35 
4.       0  .  25 
5.       0.15 
6.       0 

complete 

almost  complete 
0 

1 
0 

the  amboceptors,  that  this  binding  will  not  take  place  if  the  experi- 
ment is  performed  at  0°  C.  These  considerations  confirm  our  view 
that  the  impossibility  of  activating  the  blood-cells  sensitized  at  37°  C. 
is  due  to  a  blocking  of  the  complementophile  amboceptor  groups  of 
the  dog  serum  by  the  complementoids  of  the  same  serum. 

2.  It  still  remained  to  show  that  the  substance  which  prevented 
the  binding  subsequent  to  the  binding  effected  at  0°C.,  was  really 
present  in  the  fluid  medium.  This  could  easily  be  shown  in  the 
following  manner.  Two  parallel  series  of  tubes  with  guinea-pig  blood 
were  treated  at  0°  C.  for  one  and  one-half  hours  with  inactive  dog 
serum  (i.e.,  containing  amboceptor  +  complementoid) .  The  tubes  of 
series  A  were  then  centrifuged  and  the  sediments,  freed  from  fluid, 
suspended  in  physiological  salt  solution;  the  tubes  of  series  B  were 
left  unchanged.  All  the  tubes  were  now  placed  into  the  incubator 
for  one  hour,  then  centrifuged,  and  the  sediments  mixed  with  active 
guinea-pig  serum  and  physiological  salt  solution.  In  the  tubes  of 
series  A  solution  ensued,  the  blood-cells  of  series  B  remained  undis- 
solved,  as  can  be  seen  from  Table  III. 

The  substance  which  caused  the  blocking  of  the  ambocepters  was 
therefore  contained  in  the  fluid  portion  of  the  blood  sensitized  at  0°; 
for  in  series  A,  in  which  the  fluid  medium  was  decanted,  the  blood- 
cells  although  subsequently  kept  at  37°  C.,  could  still  be  activated. 
In  series  B,  on  the  contrary,  the  complementoids  still  remaining 
free  at  0°  C.,  were  bound  when  subsequently  kept  in  the  thermostat, 
and  so  prevented  the  "completion"  with  active  serum.  From  all 
this  it  follows  that  we  can  be  dealing  only  with  complementoid  action 
in  the  test-tube,  and  the  correctness  of  this  view  is  confirmed  in 
another  way. 


THE  MECHANISM  OF  THE  ACTION  OF  AMBOCEPTORS.     213 


TABLE  III. 


Inactive  Dog 
Serum. 

cc. 

Amount   of  Solution   of   Guinea-pig 
Blood  on  the  Addition  of  0.4  cc. 
Guinea-pig   Serum, 

Series  A. 

Series  B. 

1.        1.0 
2.       0.5 

complete 

3.       0.35 
4.       0.25 

strong 
moderate 

0 

5.       0.15 

i  f 

6.       0 

0 

3.  We  know  from  the  studies  of  v.  Dungern  1  and  Erhlich  and 
Sachs,2  that  yeast  constitutes  an  excellent  means  of  removing  the 
complements  of  a  serum.  If  we  prepared  an  inactive  dog  serum  by 
treatment  with  yeast  instead  of  with  heat,  or  if  we  allowed  the  com- 
plementoids  of  a  serum  inactivated  by  heat  to  be  absorbed  by  yeast, 
it  was  found  that  a  dog  serum  so  treated  was  no  longer  capable  of 
causing  this  "blocking"  phenomenon.  Haemolysis  occurred  in  like 
manner  whether  we  added  the  activating  guinea-pig  serum  at  once, 
or  first  kept  the  blood-cell — dog-serum  mixture  in  the  thermostat 
for  an  hour.  (See  Table  IV.) 

TABLE  IV. 


Dog  Serum, 
cc. 

Amount   of  Solution   of  Guinea-pig   Blood   on  the 
Addition  of  0.4   cc.   Guinea-pig    Serum  after 
Remaining  at  37°  C.   for    One    Hour. 

Dog  Serum  Inactivated. 

(A) 

By   Shaking   with 
Yeast.* 

(B) 
By    Heatii 
(a)  Shaken  with 
Yeast.* 

»g.  * 
(6)  Employed 
Directly. 

1.      1.0 
2.      0.5 
3.     0.35 
4.      0.25 
5.      0.15 
6.     0 

complete 

1  1 

almost  complete 
strong 
0 

complete 
« 

(  i 

almost  complete 
strong 
0 

' 

[       0 

*  6  cc.  serum  are  shaken  with  0.2  grams  yeast. 

The  complementoids  had  simply  been  removed  by  the  yeast  and  the 
isolated  amboceptors  reacted  in  normal  fashion. 


1  See  page  36  et  seq 


2  See  page  195  et  seq. 


214  COLLECTED  STUDIES   IX   IMMUNITY. 

4.  A  further  proof  of  the  correctness  of  our  view  was  furnished 
by  the  results  of  the  combining  experiment  when  the  molecular  con- 
centration of  the  fluid  medium  was  increased.  As  is  well  known  the 
haemolytic  action  of  the  sera  is  retarded  and  even  entirely  prevented 
by  an  increase  in  the  amount  of  salts  present.  'The  investigations  of 
Markl 1  have  shown  that  under  these  circumstances  the  amboceptor  is 
bound  by  the  red  blood-cells,  whereas  the  complement  is  unable  to 
take  hold.2  Through  extensive  investigations,  not  yet  published,  we 
have  been  able  to  verify  this.  Under  these  circumstances,  provided 
the  view  developed  by  us  is  correct,  it  should  naturally  be  possible 
to  prevent  the  blocking  with  complementoids  by  means  of  suitable 
concentrations  of  salts.  Two  parallel  series  of  tubes  with  guinea-pig 
blood  to  which  inactive  dog  serum  had  been  added  were  therefore 
kept  at  37°  C.  for  one  hour,  ammonium  sulphate  having  first  been 
added  to  one  of  the  series  in  the  strength  of  1.3%.  This  addition,  as 
special  tests  showed  us,  suffices  to  entirely  prevent  the  haemolytic 
.action  even  of  large  amounts  (1-  cc.)  of  active  dog  serum.  The  result 
of  the  experiment  corresponded  exactly  to  our  expectations.  The 
sediments  of  those  guinea-pig  blood-cells  which  had  been  treated 
with  ammonium  sulphate  could  be  complemented  with  guinea-pig 
serum,  whereas  in  the  other  series  no  solution  whatever  occurred. 
(See  Table  V.) 

The  analysis  of  this  case  furnishes  the  first  proof  by  means  of  test- 
tube  experiments  that  complementoids,  the  inactive  modifications  of  the 
complements,  actually  exist  in  the  inactive  serum.  To  be  sure,  even 
heretofore  their  existence  could  not  appear  doubtful,  for,  in  our 
opinion,  through  the  possibility  of  producing  antibodies,  proof  had 
been  furnished  of  the  preservation  of  the  complement's  haptophore 
group  in  the  inactivated  serum.3 

1  Markl,  Uber  Hemmung  der  Hamolyse  'durch  Salze.     Zeitschr.  f   Hygiene, 
Vol.  39,  1902. 

2  These  conditions  by  the  way,  in  our  judgment,  have  no  connection  with 
the  osmotic  conditions  of  the  cell  membrane,  as  Markl  believes.       It  seems  to 
us  that  the  action  of  the  salts  is  most  readily  explained  by  assuming  that  the 
increased  concentration  hinders  the  chemical  union  of  amboceptor  and  com- 
plement.    That  the  salts  can  exert  such  an  antireactive  action  is  seen  by  the 
fact  pointed  out  by  Knorr  (Munch  and  Wochensch.  1898,  Nos.  11  and  12)  that 
tetanus  antitoxin  and  toxin  are  absolutely  prevented  from  combining  by  the 
addition  of  10%  NaCl. 

3  In  view  of  this  new  confirmation  I  should  not  want  to  deprive  the  reader 
of  an  exposition  of  the  complementoid  theory  from  the  standpoint  of  an  opponent 


THE  MECHANISM   OF  THE  ACTION   OF  AMBOCEPTORS.     215 


TABLE  V. 


Inactive  Dog 
Serum. 

cc. 

Amount  ot  Solution  of  the  Guinea-pig  Blood 
Sediments  (Centrifuged,  after  the  Mixtures 
had  been   kept  for  One  Hour  at  37°)  on 
the  Addition  ol  0.5  cc.  Guinea-pig  Serum, 
the     Mixtures     having     been     Previously 
Treated  with 

(A) 
0.15cc.20%(NH4)2SO4 

(B) 
0.15  cc.  0.85%  NaCl. 

1.        1.0 
2.       0.5 
3.        0.35 
4.        0.25 

complete 
moderate 
little 
trace 

}     " 

In  contrast  to  the  usual  behavior  we  must  assume  that  in  the 
case  described  the  affinity  of  the  complement  has  not  suffered  any  con- 
siderable decrease  through  the  formation  of  complementoid.  This  is 
supported  also  by  an  experiment  which  we  made  in  order  to  deter- 
mine the  lowest  temperature  at  which  the  anchoring  of  the  com- 

(Proctocoll  der  k.k.  Gesellschaft  der  Aerzte  in  Wien,  Wiener  klin.  Wochenschrift, 
1901,  No.  51): 

"If  an  animal  is  injected  with  inactive  serum  of  the  same  foreign  species 
instead  of  active  serum,  it  is  found  that  its  serum  likewise  becomes  charged 
with  anticomplement ;  proof  that  the  alexin  also — like  everything  else  in  this 
world — contains  a  haptophore  group  and  an  active  group,  the  latter  this  time 
termed  zymotoxic.  As  a  result  of  the  inactivation  the  zymotoxic  group  is 
destroyed;  the  haptophore  group  remains  intact.  Hence  a  continuance  of 
the  assimilation  of  complementoid  and  the  production  of  the  anticomplement. 
So  far,  so  good.  Now,  however,  we  come  to  a  questionable  point.  If  the 
complement  deprived  of  its  zymotoxic  group  still  possesses  its  haptophore 
group,  it  must  still  be  able  to  satisfy  and  bind  its  amboceptor.  How  then 
does  it  happen  that  an  inactivated  antiserum  again  becomes  lytic  on  the  addi- 
tion of  suitable  complement  (active  normal  serum),  a  phenomenon  which, 
according  to  Ehrlich  (despite  Dr.  Wechsberg),  is  due  to  the  formation  of  lysin 
from  amboceptor  and  complement.  If  the  haptophore  group  of  the  amboceptor 
has  already  been  bound  by  the  remains  of  the  old  complement,  the  'comple- 
mentoid/ it  surely  is  unable  to  bind  new  complement,  Hence  by  heating 
(inactivating)  the  serum  the  haptophore  group  of  the  complement  cannot 
have  remained  unchanged;  it  must  have  completely  lost  its  affinity  for  the 
amboceptor.  Now,  gentlemen,  I  should  like  to  know  what  is  left  of  the  com- 
plement after  this  heating?  The  zymotoxic  group  is  destroyed,  the  haptophore 
group  so  changed  that  it  is  not  recognizable.  Nothing  remains  of  the  comple- 
ment except  Ehrlich's  fervent  wish  that  a  little  of  it  might  be  left,  because  other- 
wise it  would  not  harmonize  with  the  theory!  It  is  this  wish  that  floats  around 
in  the  inactive  serum  under  the  name  of  complementoid." 

Thus  far  Gruber!      I  shall  refrain  from  any  personal  remarks  for  which  the 


216 


COLLECTED   STUDIES  IN  IMMUNITY. 


plementoid  still  takes  place.  In  this  way  we  sought  to  find  an  approx- 
imate criterion  for  relative  affinity  of  the  complementoid.  From 
the  power  to  be  reactivated  possessed  by  the  guinea-pig  blood-cells 
previously  treated  at  different  temperatures  with  inactive  dog  serum 
it  was  seen  that  even  at  3°  C.  a  moderate  binding  of  complementoids 
takes  place,  and  that  complete  blocking  phenomena  can  already  be 
obtained  at  8°  C.,  as  is  seen  in  the  following  experiment: 

TABLE  VI. 


Inactive 
Dog  Serum. 

cc. 

Amount  of  Solution  of  Guinea-pig  Blood  on  the  Addition  of  0.5  cc. 
Guinea-pig  Serum  after  Preliminary  Treatment  at 

(A)  0°  C. 

(B)  3°C. 

(C)  6°C. 

(D)  8°  C. 

1.        1.0 
2.       0.75 
3.       0.5 
4.       0.35 

complete 
almost  complete 
strong 
moderate 

moderate 

« 

K 
little 

j-    faint  trace 

0 

Nevertheless  we  believe  that  even  in  this  case  a  certain,  though 
slight,  decrease  in  the  affinity  has  occurred  in  the  complementoid 
formation.  At  least  the  fact  speaks  in  favor  of  this,  that  with  the 
simultaneous  addition  of  inactive  dog  serum  (i.e.,  ambocep tor  +  com- 
plementoid) and  active  guinea-pig  serum  solution  of  the  guinea-pig 
blood  occurs.  Under  these  circumstances,  in  which  the  ambocep  tor 
has  both  complement  and  complementoid  to  choose  from,  the  former 
is  preferred.  When,  then,  we  find  that  it  is  possible  by  previous  treat- 
ment with  complementoid  to  block  the  complementophile  group  of 
the  ambocep  tor  for  the  complement  subsequently  added,  we  shall 
explain  this  most  readily  by  assuming  that  after  the  complementoid 
has  been  anchored,  the  union  becomes  firmer.  Analogous  phenomena 
are  common  in  immunity.  Thus  Donitz  l  has  shown  that  the  union 

unusual  tone  of  this  attack  surely  offers  sufficient  provocation,  merely  expressing 
my  astonishment  at  the  fact  that  Gruber's  exposition  disregards  the  most 
important  and  explanatory  point,  namely,  as  Morgenroth  and  I  have  emphasized, 
that  in  the  change  into  complementoids,  the  complements  must  usually  suffer  a 
decrease  in  their  affinity,  for  only  in  this  way  can  the  absence  of  all  disturbing  inter- 
ference on  the  part  of  these  complementoids  in  test-tube  experiments  be  explained. 
If,  however,  Gruber  assumes  a  complete  destruction  of  the  complements  by 
inactivation  how  does  he  explain  the  fact  easily  verified  by  every  one,  namely, 
the  production  of  anticomplements  by  injection  into  the  organism  of  serum 
which  has  been  heated?  Surely  a  mere  wish  floating  around  in  the  serum 
cannot  suffice  to  produce  anticomplements. — EHRLICH. 

1  Donitz,  tiber  die  Grenzen  der  Wirksamkeit  des  Diphtherieheilserums. 
Arch,  internat.  de  Pharmacodynam.,  Vol.  V,  1899. 


THE  MECHANISM  OF  THE  ACTION   OF  AMBOCEPTORS.     217 

of  diphtheria  poison  in  the  animal  body,  at  first  a  loose  one,  soon 
becomes  more  and  more  firm  so  that  it  cannot  be  broken  up  even 
by  very  large  amounts  of  antitoxin.  Madsen's l  experiments,  to 
liberate,  by  means  of  antitoxin,  tetanolysin  which  had  been  anchored 
by  the  blood-cells,  also  confirm  this. 

Blocking  by  means  of  complementoids  is  also  of  value  for  the 
technique  of  demonstrating  the  presence  of  amboceptor.  Suppose, 
for  example,  that  in  doubtful  cases  one  seeks  to  show  the  existence 
of  the  amboceptors  in  the  usual  manner,  by  sensitizing  the  red  blood- 
cells  and  subsequently  complementing  with  a  different  kind  of  serum. 
In  this  case,  owing  to  the  blocking  action  of  the  complementoids, 
an  absence  of  the  amboceptors  could  be  simulated.  In  this  connec- 
tion it  is  of  considerable  interest  to  know  that  so  capable  an  investi- 
gator as  Buchner2  employed  the  above  method  for  analyzing  the 
hsemolysin  in  just  the  case  here  described.  His  attempts  to  demon- 
strate the  amboceptor  by  this  method  (inapplicable  in  this  particular 
instance) ,  as  well  as  by  means  of  the  amboceptor's  thermolability a 
(already  discussed),  were  unsuccessful. 

II.  Amboceptor  or  Sensitizef  ? 

In  another  case  we  have  met  with  a  different  complication 
equally  fatal  to  the  successful  demonstration  of  the  amboceptor  by 
routine  procedures.  This  is  of  especial  interest  for  the  theory  of 
haemolysin  action,  and  concerns  the  hsemolytic  property  of  ox  serum 
for  guinea-pig  blood.  If  the  ox  serum  is  inactivated,  this  property 
can  readily  be  restored  by  the  addition  of  active  horse  serum.  If, 
however,  one  tries  by  means  of  active  horse  serum  to  complement 
blood-cell  sediments  (obtained  by  centrifuging  guinea-pig  blood  after 

1  Madsen,  Uber  Heilversuche  im  Reagensglas.     Zeitschr.  f .  Hygiene,  Vol.  32^ 
1899. 

2  H.   Buchner,    Sind   die   Alexine   einfache   oder  complexe  Korper?     Berl. 
klin.  Wochenschr.  1891,  No.  33. 

3  According  to  the  newer  researches  already  mentioned  it  would  be  con- 
ceivable that  the  thermolability  of  the  amboceptors  is  simulated  by  this, — 
that  the  complementoids,  in  themselves  possessing  a  relatively  high  affinity, 
become  firmly  anchored  to  the  amboceptors.     However,  as  special  experiments 
have  shown  us,  such  is  not  the  case,  for  dog  serum  which  has  been  inactivated 
by  shaking  with  yeast,  and  which  therefore  contains  no  complementoid,  likewise 
loses  its  ability  to  be  activated  when  it  is  heated  to  60°  C.     It  does  not  lose 
this  power  when  heated  only  to  50°-51°  C. 


218 


COLLECTED  STUDIES   IN   IMMUNITY. 


these  have  been  treated  at  37°  C.  for  one  hour  with  inactive  ox 
serum),  it  will  be  found,  just  as  in  the  previous  case,  that  haemolysis 
does  not  occur. 

The  reason  for  the  non-activatibility  in  this  case  differs  essen- 
tially from  that  in  the  case  previously  described.  The  chief  differ- 
ence manifests  itself  in  the  behavior  of  the  decanted  fluid  medium. 
If  the  centrifuging  is  omitted,  and  active  horse  serum  is  added  to 
the  sensitized  blood-cells  without  previously  removing  the  fluid 
medium,  it  will  be  found  that  solution  occurs.  If  the  centrifuging 
is  not  omitted,  it  will  be  seen  that  the  decanted  fluids  behave  in  an 
analogous  manner,  for  when  mixed  with  active  horse  serum  they 
will  dissolve  native  guinea-pig  blood.  A  complete  experiment  is 
reproduced  in  Table  VII. 

TABLE  VII. 


Inactive 
Ox  Serum. 

cc. 

Amount  of  Solution  of  Guinea-pig  Blood  on  the  Addition  of 
0.5  cc.  Horse  Serum   to  : 

(A) 
The  Sediments  on 
Centrifuging 
after  the  Mixture 
had  been  kept  at 
37°  C. 
for  One  Hour. 

(B) 

The  Decanted 
Fluids  from   (A) 
Added  to  Native 
Guinea-pig  Blood. 

(C) 
The  Uncentrifuged  Mixture  of 
Blood  and  Ox  Serum. 

(a) 
After  Remaining 
at  37°  C. 
for  One  Hour. 

(6) 
Immediately. 

1.         0.5 
2.         0.35 
3.        0.25 

4.       0.15 

5.       0.1 
6.        0 

faint  trace 

i  ;  . 

complete 

(  ( 

strong 

moderate 
0 

complete 

« 

<  t 

1      almost 
\    complete 
strong 
0 

complete 

1  1 

almost 
complete 
strong 
0 

In  contrast,  therefore,  to  the  behavior  in  the  first  case  described 
.by  us,  the  amboceptor  has  remained  in  the  decanted  fluid,  and 
has  therefore  not  been  bound  by  the  blood-cells,  or  only  to  a  very 
slight  degree.  Our  attempts  by  means,  of  horse  serum  to  activate 
the  guinea-pig  blood-cells  which  had  previously  been  treated  at 
0°  C.  with  inactive  ox  serum  and  then  centrifuged,  failed  as  a  matter 
of  course;  and  the  result  was  the  same  when  the  ox  serum  had  been 
freed  of  complementoid  by  shaking  with  yeast. 

This  peculiar  behavior,  namely,  that  the  amboceptor  by  itself 
does  not  unite  with  the  cell  at  all,  and  acts  only  after  it  has  com- 
bined with  the  complement,  is  of  special  significance  for  the  method 


THE  MECHANISM  OF  THE  ACTION  OF  AMBOCEPTORS.     219 

of  analyzing  hsemolysins.  '  For,  entirely  aside  from  the  fact  that 
under  these  circumstances  the  attempt  to  activate  the  centrifuged, 
and  presumably  "  sensitized,"  blood-cells  necessarily  fails,  it  will 
be  seen  that  the  occurrence  of  this  complication  considerably  limits 
the  application  of  the  second  method  employed  to  discover  the  com- 
plex nature  of  hsemolysins,  namely,  separation  hi  the  cold,  a  method 
already  markedly  restricted.  This  method,  it  will  be  recalled,  depends 
upon  the  fact  that  at  0°  C.  usually  only  the  amboceptors  are  bound 
to  the  blood-cells,  not  the  complements  to  the  amboceptors.  In 
the  case  just  described,  however,  the  union  of  amboceptor  and  cell 
depend  on  the  combining  of  amboceptor  and  complement.  How, 
then,  can  a  separation  of  the  two  components  be  effected  if,  on  the 
one  hand,  the  conditio  sine  qua  non  for  the  union  of  amboceptor 
and  cell,  a  condition  which  obtains  here,  cannot  be  fulfilled  at  low 
temperature,  and  if,  on  the  other,  it  in  itself  precludes  any  sepa- 
ration whatever?  No  wonder,  therefore,  that  Gruber l  failed  with 
the  cold  separation  method  in  just  this  case  (guinea-pig  blood  -f  active 
ox  serum). 

The  two  atypical  cases  here  described  are,  however,  peculiarly 
adapted  to  throw  light  on  the  mechanism  of  haemolysm  action.  In 
the  first  case  the  fact  that  blood-cells  "  sensitized  "  in  the  usual 
manner  withstand  the  action  of  the  complement  is  hard  to  explain 
in  accordance  with  Bordet's  view.  But  the  behavior  shown  in  the 
second  case  becomes  entirely  inexplicable  if,  like  Bordet,  we  believe 
the  action  of  hsemolysins  to  consist  in  this,  that  the  amboceptors 
(substance  sensibilatrice)  sensitize  the  blood-cells  and  so  render 
them  vulnerable  to  the  action  of  the  complements  (Bordet's  alexins) 
exerted  directly  upon  them.  For  here  we  have  demonstrated  that 
a  sensitization  does  not  take  place;  the  amboceptor  by  itself  is  not 
at  all  bound,  and  becomes  effective  only  on  the  addition  of  comple- 
ment. If,  however,  we  were  to  assume  that  in  our  case  the  com- 
plement nevertheless  attacks  the  cell  directly  so  that  then  the  ambo- 
ceptor can  be  found,  we  should  arrive  at  a  theory  as  unlike  Bor- 
det's as  that  held  by  Ehrlich  and  Morgenroth.  But  such  a  theory, 
strange  to  say,  would  apply  only  to  this  and  perhaps  a  few  other 
cases,  that  is,  only  to  a  few  exceptions.  Although  superfluous,  a 
suitable  experiment  was  also  made  in  this  case  and,  as  might  have 


1  Gruber,   Zur  Theorie   der  Antikorper.      Munch,   med.  Wochenschr.  1901, 
No.  49.     See  also  H.  Sachs,  1.  c. 


220  COLLECTED  STUDIES  IN  IMMUNITY. 

been  expected,  it  was  found  that  the  complement  as  such  was  not 
bound  by  the  cell. 

The  facts,  however,  are  very  readily  explained  if,  following  Ehrlich 
and  Morgenroth,  we  regard  the  amboceptor  as  a  coupler  possessing 
two  haptophore  groups.  Owing  to  a  mutual  combination  this  trans- 
mits the  action  of  the  complement  to  the  cell.  In  the  case  just 
described,  it  follows  at  once  that  the  cytophile  group  of  the  ambo- 
ceptor possesses  a  very  slight  affinity  to  the  cell  receptor.  We  have 
therefore  only  to  assume  that,  in  contrast  to  the  usual  behavior,  the 
amboceptor  in  this  case,  while  itself  unable  to  combine  with  the 
cell,  by  combining  with  the  complement  takes  on  increased  affinity 
and  so  becomes  capable  of  action. 

The  significance  of  the  variations  in  affinity  will  be  discussed 
connectedly  at ,  a  subsequent  time.  We  shall  content  ourselves 
here  by  pointing  out  that  an  understanding  of  the  phenomena  of 
immunity  is  impossible  without  the  assumption  that  certain  hapto- 
phore groups  become  increased  or  decreased  in  their  chemical  energy, 
owing  to  changes  in  the  total  molecule.  Chemically,  such  an  assump- 
tion is  a  matter  of  course.  We  believe  that  the  observations  described 
above  constitute  additional  proof  that  amboceptor  and  comple- 
ment combine  with  each  other. 

In  the  main  this  question  has  already  been  decided  by  the  beau- 
tiful investigations  of  M.  Neisser  and  Wechsberg  l  on  the  deflection 
of  complement  by  an  excess  of  amboceptor.  The  objections  raised 
against  these  experiments  by  Gruber2  and  by  Metchnikoff3  have 
been  completely  met  by  the  recent  investigations  of  Lipstein.4 

The  case  last  described  by  us  is  to  a  certain  extent  an  experi- 
mentum  crucis  for  the  correctness  of  the  views  formulated  by  Ehrlich 
and  Morgenroth  for  the  mechanism  of  hsemolysin  action.  We  there- 
fore believe  that  Bordet's  sensitization  theory  has  become  unten- 
able, and  that  now  this  question,  just  as  that  concerning  the  plurality 
of  complements,  is  definitely  closed. 

SUBSEQUENT  ADDITION. — According  to  recent  investigations  of  Dr.  Sachs,, 
guinea-pig  blood-cells,  which,  because  of  treatment  with  inactive  dog  serum, 
can  no  longer  be  dissolved  by  guinea-pig  serum,  owing  to  blocking  by  com- 

1  See  page  120  et  seq. 

2  Gruber,  Protocoll  der  k.k.  Gesellschaft  der  Aerzte  in  Wien,  Wiener  klhu 
Wochenschr.  1901,  No.  50. 

3  Metchnikoff,  PImmunit6  dans  les  malad.  infect.,  page  313,  Paris,  1901. 

4  See  page  132  et  seq. 


THE  MECHANISM  OF  THE  ACTION  OF  AMBOCEPTORS.     221 

plementoid,  are  still  dissolved  by  the  complements  of  dog  serum.  The  source 
of  the  dog  serum  complement  was  the  fluid  decanted  from  guinea-pig  blood- 
cells  which,  by  treatment  with  active  dog  serum  at  0°C.,  had  abstracted  as 
much  of  the  amboceptor  from  the  latter  as  possible.  These  experiments  there- 
fore show: 

1.  That  the  complement  of  dog  serum  suffers  a  diminution  of  affinity  in 
changing  to  complementoid. 

2.  That  the  complement  present  in  guinea-pig  serum  possesses  a  weaker 
affinity  than  the  complement  of  dog  serum  with  analogous  action. 


XX.  DIFFERENTIATING  COMPLEMENTS  BY  MEANS  OF 
A  PARTIAL  ANTICOMPLEMENT.1 

By  H.  T.  MARSHALL,  Fellow  of  the  Rockefeller  Institute,  and  Dr.  J.  MORGEN- 
ROTH,  Member  of  the  Frankfurt  Institute. 

THE  question  whether  the  serum  of  one  and  the  same  species 
contains  a  plurality  of  complements  or  only  a  single  one  seems  to 
us  to  have  been  positively  decided  in  favor  of  the  pluralistic  concep- 
tion. This  decision  has  been  brought  about  mainly  by  the  observa- 
tions of  Ehrlich  and  Morgenroth,2  of  Wassermann,3  Wechsberg,4 
Wendelstadt,5  and  by  the  recently  published  studies  of  Ehrlich  and 
Sachs.6  Nevertheless,  we  shall  briefly  describe  an  experiment  which, 
in  a  single  instance  at  least,  constitutes  a  proof  for  the  plurality  of 
the  complements.  Our  object  in  doing  this  is  not  that  the  number 
of  arguments  may  be  further  increased,  for  they  are  already  amply 
sufficient,  but  that  we  may  call  attention  to  a  method  of  demonstra- 
tion which  has  not  heretofore  been  employed. 

Because  of  purely  technical  difficulties  the  most  rational  and 
simplest  method  of  differentiation,  namely,  by  means  of  anticomple- 
ments,  has  not  thus  far  been  employed  in  this  question.  As  is  well 
known,  it  is  very  easy  by  immunizing  with  serum  containing  com- 
plement or  complementoid  to  obtain  potent  anticomplements.  Such 
anticomplejnent  sera,  however,  usually  contain  anticomplements  cor- 
responding to  the  sum  of  all  the  complements  originally  injected,7 
and  are,  therefore,  not  adapted  to  the  separation  of  complements. 

1  Reprinted  from  the  Centralblatt  f.  Bact.       Original  Vol.  XXXI,  No.  12, 
1902. 

2  See  pages  11,  56,  86. 

3  Wassermann,   Zeitschr.  f.  Hyg.,  Vol.  XXXVII,    1901. 

4  Wechsberg,  Sitzung  d.  k.  k.  Ges.  d.  Aerzte  in  Wien,  Wiener  klin.  Wochen- 
schr.  1901,  No.  48. 

6  Wendelstadt,  Centralblatt  f.  Bact.     Part  I,  Vol.  XXXI,  No.  10. 
e  See  page  195  et  seq. 
1  See  page  63  et  seq. 

222 


DIFFERENTIATING  COMPLEMENTS.  223 

At  least  this  had  been  the  case  thus  far,  for  a  partial  anticomplement, 
one  acting  only  against  a  single  complement,  had  not  been  observed. 

Through  the  courtesy  of  Dr.  Cnyrim  we  detained  a  normal  anti- 
complement  which  possessed  the  desired  properties,  and  we  there- 
fore gladly  availed  ourselves  of  this  favorable  opportunity  to  demon- 
strate, by  means  of  the  elective  binding  of  anticomplement,  the 
difference  between  two  complements  in  one  and  the  same  serum,  a 
difference  that  had  not  heretofore  been  demonstrated.1 

This  normal  anticomplement  was  an  ascitic  fluid  derived  from  a 
case  of  cirrhosis  of  the  liver;  it  exerted  a  marked  antihffimolytic 
action  in  one  particular  case.  By  means  of  an  experiment  we  first 
determined  that  this  action  was  due  to  the  presence  of  an  anticomple- 
ment and  not  of  an  anti-immune  body.  This  showed  us  that  the 
ascitic  fluid  exerted  practically  no  influence  on  the  anchoring  of  the 
immune  body  in  question  to  the  red  blood-cells. 

The  serum  whose  complements  we  examined  was  guinea-pig 
serum,  which  activated  two  amboceptors  obtained  by  immunization. 
These  amboceptors  were  contained  in  the  inactive  serum  of  a  rabbit, 
A,  which  had  been  immunized  with  ox  blood;  and  in  the  inactive 
serum  of  a  goat,  B,  which  had  been  immunized  with  sheep  blood. 
Corresponding  to  this,  ox  blood  was  used  for  case  A,  and  sheep  blood 
for  case  B.  The  inactive  ascitic  fluid  does  not  dissolve  these  species 
of  blood  even  after  the  addition  of  guinea-pig  serum 

To  begin,  we  saturated  ox  blood-cells  with  the  specific  amboceptor 
by  adding  0.01  cc.  immune  body  A  to  each  1  cc.  of  a  5%  suspension 
of  the  cells.  This  is  about  ten  times  the  amount  which  on  the  addi- 
tion of  sufficient  complement  (0.1  cc.  guinea-pig  serum)  effected 
complete  solution.  The  mixture  was  placed  in  the  incubator  and 
frequently  shaken.  At  the  end  of  one  hour  it  was  centrifuged,  the 
fluid  poured  off,  and  the  blood-cells,  loaded  with  amboceptor,  sus- 
pended in  salt  solution.  In  exactly  the  same  manner  sheep  blood- 
cells  were  treated  with  the  inactive  serum  B,  0.2  cc.  for  each  1  cc. 
of  the  5%  suspension.  On  the  addition  of  guinea-pig  serum  to  these 
blood-cells,  hsmolysis  ensued  very  quickly  in  the  thermostat;  in 
both  cases  it  required  0.008  cc.  guinea-pig  serum  to  fully  dissolve  1  cc.  of 
the  suspension,  while  0.0065  cc.  caused  incomplete  solution  and  0.002 

1  In  the  following,  for  the  sake  of  simplicity,  we  shall  speak  only  of  two 
complements,  whereas  we  wish  here  to  remark  that  two  groups  of  complements 
are  probably  to  be  understood,  each  group  made  up  of  a  host  of  single  comple- 
ments which  it  is  impossible  thus  far  to  analyze. 


224 


COLLECTED   STUDIES  IN   IMMUNITY. 


only  a  slight  degree  of  solution.  For  the  sake  of  clearness  it  was 
especially  fortunate  that  the  complementing  amounts  should  happen 
to  be  identical  in  the  two  cases. 

A  parallel  series  of  experiments  was  then  undertaken  with  these 
two  cases,  as  follows:  Varying  amounts  of  the  guinea-pig  serum  were 
mixed  each  with  0.4  cc.  of  ascitic  fluid  inactivated  at  56°  C.,  and 
the  mixtures  kept  at  room  temperature  for  half  an  hour,  after  which 
the  binding  was  entirely  completed.1  Thereupon  the  blood-cells 
loaded  with  amboceptor  were  added.  The  result  of  these  experiments 
is  shown  in  the  following  table : 

CASE  A  (Ox  BLOOD + AMBOCEPTOR). 


Guinea-pig  Serum  Alone. 

Guinea-pig    Serum  +0.4   cc. 
Ascitic    Fluid. 

0.008    complete  solution 
0.0065  vestige 
0.005    strong 
0  .  0055  considerable 
0.003 
0.0025  moderate 

0.1      almost  complete 
0.08 
0  .  065  considerable 
0.05    fairly  little 
0.035  very  little 
0.03    trace 
0.025     " 
0.02        0 

CASE  B  (SHEEP  BLOOD  +  AMBOCEPTOR). 


Guinea-pig  Serum  Alone. 

Guinea-pig  Serum  +  0.4  cc. 
Ascitic  Fluid. 

0.008    complete  solution 
0  .  0065  almost  complete 
0.005 
0.0035  strong 

0.008    complete 
0  .  0065  almost  complete 
0.005 
0.0035  strong 

We  see,  therefore,  that  in  case  A  the  complement  protects  com- 
pletely against  2J  times  the  complete  solvent  amount  of  complement, 
while  the  amount  of  serum  required  to  effect  complete  solution  increases 
more  than  twelve  times.  In  case  B,  on  the  contrary,  the  complete 
solvent  dose  of  guinea-pig  serum  remains  unchanged,  and  the  series 
proceeds  just  as  though  there  had  been  no  addition  of  antieomple- 
ment. 

These  experiments,  which  were  repeated  many  times,  therefore 

1  The  union  of  complements  and  anticomplements,  analogous  to  the  behavior 
of  certain  toxins  and  antitoxins,  is  dependent  on  the  time.  Hence  here  also 
this  had  to  be  considered  and  sufficient  time  allowed  for  the  mixture  to  act 


DIFFERENTIATING  COMPLEMENTS.  225 

show  that  the  ascitic  fluid  contains  an  anticomplement l  which  fits 
into  that  complement  which  is  activated  by  amboceptor  A,  whereas 
anticomplements  for  the  complement  of  amboceptor  B  are  absent. 
Hence  we  are  justified  in  differentiating  in  guinea-pig  serum  at  least 
two  complements  with  different  haptophore  groups. 

It  may  be  hoped  that  continued  investigations  of  normal  body 
fluids  will  bring  to  light  numerous  other  favorable  cases  which  will 
make  possible  differences  along  the  lines  indicated.  For  although 
in  normal  serum  the  complication  of  haptins  present,  such  as  ambo- 
ceptors,  complements,  complementoids,  antiamboceptors,  and  anti- 
complements,  is  very  great,  the  conditions  here  are  certainly  simpler 
than  in  the  serum  of  immunized  animals;  for  in  the  latter  there  are 
also  present  innumerable  primary,  and  (owing  to  internal  regulative 
processes)  secondary  reactive  products. 

1  Erhlich  and  Morgenroth  have  discussed  the  nature  of  anticomplements 
at  length  in  the  Berl.  klin.  Wochenschr.  1901,  No.  10.  They  conclude  that 
the  origin  of  these  bodies  is  this,  that  foreign  complements  combine  with  the 
complementophile  group  of  certain  cell  receptors.  According  to  this  view 
the  anticomplements  are  nothing  else  than  thrust-off  ambocepters  whose  com- 
plementophile groups  possess  a  higher  affinity  than  is  usually  the  case.  It  is 
curious,  therefore,  that  Gruber,  nine  months  later  (Sitzg.  der  k.k.  Ges.  der 
Aerzte  in  Wien,  Wiener  klin.  Wochenschr.  1901),  presents  this  view,  which 
had  been  recognized  as  a  natural  consequence  of  the  receptor  theory,  as  an 
entirely  new  objection  against  just  this  theory. 


XXI.  CONCERNING  THE  COMPLEMENTOPHILE  GROUPS 
OF  THE  AMBOCEPTORS.1 


By  Prof.  Dr.  P.  EHRLICH  and  H.  T.  MARSHALL,  M.D.,  Fellow  of  the  Rockefeller 
Institute  of  Medical  Research. 


THE  studies  of  the  past  year,  especially  the  recent  conclusive 
work  of  Ehrlich  and  Sachs,2  show  that  we  may  regard  it  as  definitely 
proven  that,  in  contrast  to  the  Unitarian  conception  of  Bordet,  there 
is  a  plurality  of  complements  in  the  serum. 

This  knowledge  largely  supplements  our  views  concerning  the 
mechanism  of  lysin  action,  and  is  in  complete  harmony  with  the 
principles  of  the  amboceptor  theory.  The  latter,  in  contrast  to  the 
untenable  sensitization  theory  of  Bordet,  has  become  still  more 
firmly  established  through  the  recent  experiments  carried  out  in  the 
Institute  by  M.  Neisser  and  Wechsberg,3  Lipstein,4  and  Ehrlich  and 
Sachs.5 

If  we  consider  that,  as  is  shown  especially  by  Bordet's  experi- 
ments,6 an  amboceptor,  after  having  been  anchored  by  cellular  ele- 
ments, can  almost  completely  rob  a  serum  of  its  complement,  and  if, 
further,  we  regard  what  we  now  know  about  the  plurality  of  comple- 
ments, we  shall  of  necessity  be  led  to  a  view  concerning  amboceptors 
according  to  which  an  amboceptor  is  capable  of  binding  a  number  of 
different  complements  simultaneously.  Attention  was  called  to  such 
a  possibility  by  Ehrlich  and  Morgenroth  7  when  they  stated :  "  Finally, 
it  is  possible  that  an  immune  body,  besides  one  particular  cytophile 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  No.  25. 

2  See  page  195.         3  See  page  120.         *  See  page  132.         B  See  page  209. 
•  Bordet,  AnnaL  de  1'Institut  Pasteur,  May  1901. 

7  See  pages  88  et  seq. 

226 


COMPLEMENTOPHILE  GROUPS  OF  THE  AMBOCEPTORS.     227 

group,  contains  two,  three,  or  more  complementophile  groups." 
According  to  this  latter  view,  therefore,  it  is  to  be  assumed  that  an 
amboceptor  possesses  one  haptophore  group  specifically  related  to  a 
certain  receptor  of  cell  or  of  a  foodstuff,  and  that  it  also  possesses  a 
number  of  complementophile  groups.  The  term  amboceptor  would 
thus  indicate  that  two  different  substances,  foodstuff  and  comple- 
ment, are  anchored  by  this  body  and  brought  into  close  relation  with 
each  other.  This  is  illustrated  in  the  following  diagram. 

FIG.  l. 


(a)  receptor  of  the  cell;  (6)  haptophore  group  of  the  amboceptor;  (c)  domi- 
nant complement;  (d)  non-dominant  complement. 

Complementophile  groups  of  the  amboceptor:  (a)  for  the  dominant  com- 
plement; (/?)  for  the  non-dominant. 

The  next  question  to  be  considered  is  whether  it  is  necessary,  in 
order  to  get  the  specific  lysin  effect,  for  all  these  complements  to  come 
into  action.  Recent  experiments  indicate  that  this  is  not  the  case, 
but  that  among  the  number  of  complements  only  a  few  are  necessary 
in  any  single  instance  in  order  to  obtain  the  effect.  These  comple- 
ments are  termed  "dominant  complements"  the  rest  are  "non-dominant 
complements." 


228  COLLECTED  STUDIES  IN  IMMUNITY. 

A  case  described  by  Ehrlich  and  Sachs  makes  this  clear,  and 
we  shall  therefore  briefly  reproduce  it  here: l 

Two  amboceptors  are  concerned,  namely,  the  normal  ambocep- 
tor  of  goat  serum  for  rabbit  blood,  and  an  amboceptor  obtained 
by  immunizing  goats,  which  is  anchored  by  ox  blood-cells.  We 
shall  for  the  sake  of  simplicity  designate  these  amboceptors  as  A 
and  B. 

Naturally  both  these  amboceptors  are  activated  by  goat  serum, 
in  which  we  shall  have  to  assume  at  least  two  complements  x  and  b. 
For  immune  body  A,  x  is  the  dominant  complement;  for  B  it  is  6. 
If  in  one  of  the  two  combinations,  for  example,  in  that  of  rabbit 
blood-cells  loaded  with  immune  body  A,  the  serum  is  allowed  to  act 
long  enough,  both  complements  will  be  bound;  that  is,  dominant 
and  non-dominant.  The  result,  however,  is  entirely  different  if  the 
action  be  made  as  short  as  possible.  In  this  case  the  fluid  obtained 
on  centrifuging  the  blood-cells  still  contains  the  dominant  comple- 
ment x,  while  it  has  for  the  most  part  lost  the  non-dominant  com- 
plement 6.  We  observe  the  surprising  result  that  the  immune  body 
A  with  which  the  blood-cells  are  loaded  combines  with  the  non- 
dominant  complement  before  it  combines  with  its  own  dominant 
complement. 

In  this  case,  therefore,  amboceptor  A's  complementophile  groups 
which  combine  with  the  complement  must  possess  a  higher  affinity 
for  the  non-dominant  complement  b  than  for  the  dominant  comple- 
ment x.  Here  then  the  binding  of  the  non-dominant  complement  is 
independent  of  the  binding  of  the  dominant  complement.  Such  a 
behavior,  of  course,  is  not  a  general  rule;  it  was  not  long  before 
a  case  was  found  in  which  the  contrary  was  true,  i.e.,  in  which  the 
non-dominant  complement  does  not  combine  until  after  the  dominant 
complement  has  been  bound. 

.  The  demonstration  of  this  relation  succeeded  only  because  in  a 
certain  human  ascitic  fluid  an  anticomplement  was  present  whfch 
acted  only  against  part  of  the  complements  of  a  serum.  The  peculiar 
behavior  of  this  anticomplement  has  been  described  in  a  recent  com- 
munication by  Marshall  and  Morgenroth,2  and  is  also  readily  seen 
in  the  following  experiment.  The  complements  here  concerned  are 


1  For  the  sake  of  clearness  the  case  has  here  been  somewhat  simplified.    The 
details  of  this  experiment  are  found  in  Ehrlich  and  Sachs,  page  195. 

2  See  page  222. 


COMPLEMENTOPHILE  GROUPS  OF  THE  AMBOCEPTORS.    229 

present  in  normal  guinea-pig  serum.  This  serum  reactivates  two 
immune  bodies,  of  which  one,  immune  body  A,  was  obtained  by  treat- 
ing rabbits  with  ox  blood,  and  the  other,  immune  body  B,  by  treat- 
ing a  goat  with  sheep  blood.  These  immune  bodies,  naturally,  acted 
respectively  on  ox  blood-cells  and  sheep  blood-cells.  This  anti- 
complement  is  strongly  active  in  case  A,  while  it  is  entirely  without 
effect  in  case  B.  From  this  we  may  conclude  that  the  complements 
concerned  in  these  two  cases,  and  which  we  may  designate  as  x  and 
6,  are  unlike. 

A  further  question  was  whether  immune  body  A  binds  other  com- 
plements in  guinea-pig  serum  besides  its  own  dominant  complement. 
In  order  to  determine  this  the  following  experiment  was  made:  First, 
ox  blood-cells  and  sheep  blood-cells  were  saturated  with  their  re- 
spective amboceptors  A  and  B,  and  then  to  each  cubic  centimeter  of 
the  5%  blood  suspension  varying  amounts  of  guinea-pig  serum  were 
added  as  complement.  In  the  first  case  0.0075  cc.  guinea-pig  serum 
sufficed  to  cause  complete  solution;  in  the  second  case  0.005  cc. 
was  required. 

Thereupon  another  test  was  made  exactly  like  the  preceding  with 
ox  blood  and  immune  body  A.  After  the  mixture  had  remained  in 
the  thermostat  at  37°  C.  for  1^  hours  and  haemolysis  was  practically 
completed,  the  same  quantity  of  ox  blood-cells  laden  with  immune 
body  (0.05  cc.  ox  blood  freed  from  serum  and  made  up  to  the  original 
volume)  was  added  anew  and  the  mixtures  kept  in  the  thermostat 
for  two  hours  longer.  The  haemolysis  which  had  then  taken  place, 
observed  by  allowing  the  mixture  to  sediment  in  a  refrigerator,  indi- 
cated the  amount  of  complement  x  left  after  the  first  haemolysis  and 
available  for  the  case  A. 

At  the  same  time  a  similar  experiment  was  made  in  which, 
after  the  first  haemolysis,  sheep  blood-cells  saturated  with  ambo- 
ceptor  were  used  in  the  place  of  the  ox  blood-cells.  In  this  case, 
after  determining  the  amount  of  complement  originally  present, 
that  of  complement  6,  left  after  the  first  haemolysis,  could  also  be 
found. 

In  this  a  considerable  loss  of  complement  is  observed  for  both 
cases;  for  it  now  requires  0.075  cc.  of  the  complementing  guinea- 
pig  serum  to  cause  complete  solution  for  case  A  and  0.025  cc.  for 
case  B,  so  that  1/10  and  1/5  respectively  of  the  original  complement 
are  still  preserved.  This  shows  that  the  binding  of  complement  a, 
dominant  for  case  A,  is  accompanied  by  a  binding  of  complement/?, 


230 


COLLECTED  STUDIES  IN  IMMUNITY. 


dominant  for  case  B  but  non-dominant  for  case  A.  It  was  next 
necessary  to  determine  whether  or  not  in  case  A  the  absorption  of 
the  non-dominant  complement  ft  is  dependent  on  the  binding  of  the 
dominant  complement  a.  Owing  to  the  peculiar  nature  of  the  anti- 
complement  it  is  possible  to  prevent  the  binding  of  complement  a 
for  case  A,  whereas  the  binding  of  complement  ft  for  case  B  is 
not  affected.  On  the  addition  of  0.4  cc.  of  the  anticomplement 
serum  the  amount  of  complement  necessary  for  complete  solution 
increases  from  0.0075  cc.  to  0.2  cc.,  i.e.  26  times,  whereas  no  change 
occurs  for  case  B,  0.005  of  the  guinea-pig  serum  still  causing  com- 
plete solution. 

If,  therefore,  the  binding  of  the  complement  ft  by  ox  blood-cells 
laden  with  amboceptor  A  is  dependent  on  the  binding  of  the  domi- 
nant, complement  a,  it  must  be  possible  by  the  addition  of  the  fluid 
containing  the  anticomplement  to  prevent  this  binding.  The  ex- 
periment is  made  'as  follows : 

First,  0.4  cc.  anticomplement  serum  is  mixed  with  varying 
amounts  of  guinea-pig  serum.  After  this  mixture  has  remained  at 
room  temperature  for  half  an  hour  the  ox  blood-cells  laden  with 
amboceptor  are  added  and  the  whole  kept  in  the  thermostat  for 
1J  hours,  when  the  undissolved  blood-cells  are  centrifuged  off.  The 
decanted  fluid  is  mixed  sheep  blood-cells  loaded  with  their  ambo- 
ceptor. The  result  shows  that  in  this  case  a  decrease  of  complement 
b  for  B  has  not  occurred,  for  the  tube  containing  0.005  guinea-pig 
serum  shows  complete  solution.  The  following  table  will  make  the 
results  plain: 

COMPLETE  SOLVENT  AMOUNTS  OF  GUINEA-PIG  SERUM. 


I. 

II. 

III. 

IV. 

Absolute     Deter- 

After Binding  the 

After  Binding  the 

Amount    of   Com- 

mination of  the 
Complement. 

Complement  by 
Means  of  Ambo- 

Complement by 
Means  of  0.4  cc. 

plement  Used  by 
Amboceptor     + 

ceptor  -f-  Blood- 
cells  (Case  A). 

Anticomplement 

Blood-cells  (Case 
A  )  after  Binding 

of  the  dominant 

Complement    by 

Means  of  0.4  cc. 

Anticomplement 

On  c*f\      A 

0007^ 

Of)7K 

09 

v^ase  A.  

.  UU  i  O 

.  u  to 

.  4 

Case  B 

0.005 

0.025 

0.005 

0.005 

By  means  of  this  experiment,  therefore,  it  has  been  proved  that 
in  this  case  binding  of  the  non-dominant  complement  ensues  only  after 
the  corresponding  complementophile  group  of  immune  body  A  has  an- 


COMPLEMENTOPHILE  GROUPS  OF  THE  AMBOCEPTORS.    231 

chored  the  dominant  complement  a.  We  shall  probably  not  be  wrong 
if  we  assume  that  in  this  case,  owing  to  the  occupation  of  the  com- 
plementophile  group  for  a,  there  is  an  increase  in  affinity  of  the  com- 
plementophile  group  for  /?.  The  subject  of  hsemolysins  contains 
many  analogies  for  such  a  behavior.  Thus  it  is  quite  common  that 
not  until  the  haptophore  group  of  an  amboceptor  is  bound  to  a  cell 
does  the  complementophile  group  of  the  same  possess  sufficient 
affinity  to  anchor  the  complement. 

Such  an  arrangement,  whereby  a  single  amboceptor  is  able  to 
bind  a  number  of  different  complements,  is  certainly  not  useless. 
Owing  to  their  zymotoxic  groups  the  complements  can  manifestly 
exercise  quite  different  actions,  so  that  the  digestion  of  highly  com- 
plex food  molecules — in  which,  of  course,  we  must  see  the  physiological 
function  of  the  amboceptor  mechanism — is  surely  made  easier.  Such 
an  arrangement  seems  still  more  adapted  to  the  purpose  when  we 
consider  that  the  cytophilic  haptophore  group  of  an  amboceptor  is 
fitted,  not  to  the  entire  food  molecule  as  such,  but  only  to  a  partial 
group  of  the  food  molecule.  The  possibility  is  thus  given  for  a  par- 
ticular amboceptor  to  anchor  foodstuffs,  which  are  .almost  entirely 
different  but  happen  to  agree  in  the  possession  of  this  one  partial 
group.  Granted  that  this  is  the  case,  the  presence  of  only  a  single 
complement,  acting  only  in  one  or  the  other  possibility,  would  be 
clysteleological,  whereas  a  plurality  of  complements  would  insure  the 
greatest  possible  effect  on  the  most  varied  foodstuff  molecules.  Re- 
cent investigations  have  brought  to  light  a  great  many  examples 
which  show  that  in  extracellular  and  intracellular  digestive  processes 
various  ferments  act  together  or  in  sequence.  Thus,  as  Hofmeister l 
states,  we  already  know  of  ten  different  ferments  in  the  liver-cell: 
"A  maltase,  a  glycase,  a  proteolytic  ferment,  a  nuclein-splitting 
ferment,  an  aldehydase,  a  lactase,  a  ferment  which  converts  the 
firmly  bound  nitrogen  of  amido  acids  into  ammonia,  a  fibrin  ferment, 
and,  with  some  probability,  a  lipase  and  a  rennin-like  ferment."  Even 
in  so  simple  an  organism  as  the  yeast-cell,  according  to  Delbriick,2 
at  least  five  endoferments  are  demonstrable. 

If  one  cares,  one  can  regard  an  amboceptor  whose  various  comple- 
mentophile groups  are  occupied  by  different  complements  as  a  kind 


1  Hofmeister,  Die  chemische  Organisation  der  Zelle.    Vortrag.    Braunschweig, 
1901. 

2  Delbriick,  Jahrbuch  des  Vereins  der  Spirit usfabrikanten,  Vol.  II,  1902. 


232  COLLECTED  STUDIES  IN  IMMUNITY. 

of  poly  enzyme.  Analogous  views  have  been  expressed  by  Nencki l 
for  the  ferments  of  the  digestive  tract.  Even  though  his  conception, 
that  pepsin  is  a  single  ferment  with  different  active  groups  (pepsin 
group,  rennin  group,  plastin-forming  group),  does  not  entirely  apply, 
we  must  say  that  his  conception  of  such  polyenzymes  is  fully  justi- 
fied. The  properties  of  the  amboceptor  above  demonstrated  will,  we 
believe,  speak  in  favor  of  the  essential  soundness  of  the  view  of  this 
eminent  chemist. 

1  Nencki  and  Sieber,  Zeitschr.  f .  physiol.  Chem.  1901. 


XXII.    CONCERNING  THE   COMPLEMENTIBILITY  OF 
THE  AMBOCEPTORS.1 

By  Dr.  J.  MORGENROTH,  Member  of  the  Institute,  and  Dr.  H.  SACHS,  Assistant 

at  the  Institute. 

I.  A  Presumptive  Law  Concerning  the  Complementibility  of  Normal 
Amboceptors  and  those  Obtained  by  Immunization. 

GRUBER2  believes  he  has  discovered  an  essential  difference  in  the 
Complementibility  of  the  normal  amboceptors  of  blood  serum  and 
those  produced  by  immunization.  He  says:  "The  amboceptors3 
of  the  normal  sera  never  seem  to  make  the  erythrocytes  of  another 
species  sensitive  to  their  own  serum,  .  .  .  and  I  think  I  can  say  before- 
hand that  the  specific  amboceptors  regularly  make  the  erythrocytes 
soluble  in  their  own  serum.  This  would  constitute  an  essential 
difference  between  the  two." 

If  Gruber  believes  Ehrlich  has  ever  maintained  that  the  ambo- 
ceptors of  normal  and  of  immune  sera  are  identical,  this  is  a  mis- 
understanding. On  the  contrary,  the  studies  at  this  Institute  4  have 
emphasized  that  the  immune  sera,  owing  to  the  manifold  variety 
of  the  reaction  products  developed  in  the  immunization,  contain  a 
great  host  of  different  partial  amboceptors  whose  cytophile  and 
complementophile  groups  can  vary  greatly.  Normal  serum,  on  the 
contrary,  possesses  only  few  types  of  amboceptors  identical  with 
those  of  the  immune  serum.  Hence  if  there  is  to  be  any  question  at 
all  as  to  the  identity  of  normal  and  artificially  produced  amboceptors, 
this  can  only  be  a  partial  identity.  Special  proof  by  Gruber  of  their 
non-identity  in  order  to  controvert  the  opposite  view  was  therefore 
unnecessary.  However,  since  what  Gruber  advances  is  incorrect  and  in 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  No.  27. 

2  Gruber,  Munch,  med.  Wochenschr.  1901,  No.  49. 
8  Gruber  terms  this  "  preparator." 

4  See  especially  pages  88  et  seq. 

233 


234 


COLLECTED   STUDIES  IN   IMMUNITY. 


contradiction  to  our  experimental  results,  let  us  examine  his  evidence 
somewhat  more  closely. 

In  support  of  his  first  assertion,  that  "  the  amboceptor  of  the 
normal  sera  seems  never  to  make  the  erythrocytes  of  another  species 
sensitive  to  their  own  serum,"  he  advances  the  following  eight  com- 
binations (see  Table  I) : 

TABLE  I. 


Number. 

Species  of  Blood  and 
Complement. 

Amboceptor. 

1 

rabbit 

OX 

2 
3 

guinea-pig 
rabbit 

(  ( 

dog 

4 

gumea-pig 

1  ' 

5 

6 

i  ( 

sheep 
rabbit 

7 

1  < 

chicken 

8 

rabbit 

sheep 

It  seems  entirely  to  have  escaped  Gruber  that  only  a  few 
lines  previously  he  denies  the  existence  of  amboceptor  in  the  first 
three  of  these  combinations.  Hence  he  should  not  have  included 
these  as  evidences  of  the  amboceptor's  non-activatibility.  for  his 
own  experiments  had  shown  him  that  in  these  ha?molysins  no  ambo- 
ceptors could  have  been  present.1  From  our  own  experiments  we 
know  that  the  next  three  combinations  (4-6)  usually  lead  to  solution 
of  the  blood;  there  remain  therefore  only  two  cases  (7  and  8)  which 
we  may  consider  as  evidence  of  Gruber's  contention.2  Against  these 
two  cases  can  be  placed  a  single  case  described  by  Gruber,  one  which 
he  advances  to  support  his  second  statement,  "that  the  specific 
amboceptors  make  the  erythro  ytes  soluble  in  their  own  serum." 
Gruber  believes  he  can  say  in  advance  that  this  is  regularly  the  case. 

1  Since  then,  however,  amboceptors  have  also  been  demonstrated  in  these 
cases.     (See  H.  Sachs,  page  181.) 

2  One  of  these  cases  deals  with  the  combination  guinea-pig  blood  +  chicken 
serum.     From  Ehrlich  and  Morgenroth's  earlier  communications  (see  pages  88 
et  seq.)  Gruber  could  have  seen  that  between  animal  species  so  far  removed 
as  chicken  and  guinea-pig  the  chances  of  complementibility  are  not  as  great 
as  they  are  between  mammalian  species.     If  Gruber  therefore  employs  as  evi- 
dence such  distantly  related  species  he  must  necessarily  also  have  used  widely 
separated  species  when  complementing  the  immune  sera.     We  have  no  doubt 
at  all  that  by  immunizing  distantly  related  species  (birds)   with  guinea-pig 
blood,  amboceptors  can  be  obtained  which  are  not  complemented  by  guinea- 
pig  serum,  or  at  least  not  regularly  so. 


COMPLEMENTIB1L1TY  OF  THE  AMBOCEPTORS. 


235 


Gruber  has  prophesied  correctly.  To  one  who  has  familiarized 
himself  with  the  plurality  of  the  amboceptors  it  will,  to  be  sure, 
appear  a  matter  of  course  that  the  erythrocytes  loaded  with  specific 
amboceptors  usually  find  suitable  complements  which  cause  their 
solution,  as  in  most  other  sera,  so  also  in  their  own  serum.  As  a 
matter  of  fact,  according  to  our  own  experience,  the  amboceptors  of 
the  immune  sera  seem  as  a  rule  to  make  the  blood-cells  sensitive  to 
their  own  serum.  But  the  far-reaching  difference  between  the  im- 
mune sera  and  normal  sera  which  Gruber  sees  in  this  fact  does  not 
exist. 

In  the  following  table  we  have  collected,  either  from  personal 
knowledge  or  from  the  statements  of  other  authors,1  those  cases  in 
which  the  combinations  blood-cells  a  +  inactive  normal  serum  (am- 
boceptor)  6  + complement  a  lead  to  haemolysis,  in  contradiction  to  their 
behavior  as  stated  by  Gruber.  (See  Table  II.) 

TABLE  II. 


Number. 

Species  of  Blood  and 
of  Complement. 

Amboceptor. 

1 
2 

guinea-pig 

dog 
calf 

3 

goat 

rabbit 

4 

sheep 

1                    U 

5 

guinea-pig 

sheep 

6 

l( 

horse 

7 

t  e 

ox 

8 

rabbit 

" 

9 

t  c 

man 

10 

guinea-pig 

rabbit 

This  table,  which  makes  no  pretense  at  completeness,  shows  that 
the  solubility,  in  their  own  serum,  of  blood-cells  loaded  with  normal 
amboceptor  is  quite  common.  This  becomes  still  more  evident 
when  we  consider  that  the  combinations  mentioned  include  only  a 
limited  number  of  the  most  common  experimental  animals,  and  that 
by  using  other  species  still  more  combinations  would  be  found. 

Gruber's  statements  therefore  are  all  the  more  surprising  since  a 
large  part  of  the  cases  here  reproduced  have  already  been  described 
in  the  literature.  Just  this  activatibility  of  normal  amboceptors 

1  Erhlich  and  Morgenroth,  page  11 ;  Xeisser  and  Doring,  Berl.  klin.  Wochen- 
schr.  1901,  No.  22;  H.  Buchner,  Berl.  klin.  Wochenschr.  1901,  No.  33;  H. 
Sachs,  page  181. 


236  COLLECTED  STUDIES  IN  IMMUNITY. 

by  means  of  serum  corresponding  to  the  blood-cells  employed  has 
very  recently  been  employed  by  Buchner  1  exclusively  as  a  reaction 
for  the  presence  of  normal  amboceptors. 

Although  the  principle  advanced  by  Gruber  as  an  invariable 
means  of  differentiation  has  failed,  we  are  far  from  identifying  normal 
and  specific  amboceptors.  As  already  stated,  we  believe  that  in  the 
sense  above  described  it  has  been  proved  that  they  vary.  Here  we 
should  like  to  emphasize  that,  despite  individual  multiplicity,  all 
amboceptors  belong  essentially  to  a  common  class  of  similarly  react- 
ing substances. 

To  us  these  observations  appear  of  interest  also  in  another  direc- 
tion. Baumgarten2  ascribes  the  haemolysis  in  a  foreign  serum 
entirely  to  the  influence  of  the  amboceptors,  which  he  identifies  with 
the  agglutinins.  He  says  that  "while  in  themselves  incapable  of 
effecting  haemolysis,  they  put  the  red  blood-cells  into  such  a  condition 
that  they  allow  their  haemoglobin  to  escape  even  on  relatively  slight 
osmotic  disturbances."  Just  these  slight  osmotic  disturbances, 
according  to  Baumgarten,  are  caused  by  the  foreign  sera  whose 
osmotic  tension  is  changed  by  heating  (inactivation).  Hence  Baum- 
garten regards  the  assumption  of  complements  as  entirely  unnecessary. 
In  opposition  to  this  we  would  like  to  call  to  mind  the  numerous 
combinations  described  by  us  (even  Bordet  has  described  such  for 
the  haemolysins  obtained  by  immunization),  in  which  the  blood- 
cells  dissolve  in  their  own  serum,,  i.e.  in  the  ideal  isotonic  medium, 
if  they  have  previously  been  treated  with  an  inactive  serum  (ambo- 
ceptor)  of  a  different  species.  Such  cases  clearly  indicate  that  ha> 
molysis  by  means  of  blood  serum  has  nothing  to  do  with  isotonic 
conditions;  that  it  is  rather  due  to  a  poisonous  action  which  depends 
on  the  coaction  of  two  components — amboceptor  and  complement. 

II.    Concerning  the  Variability  of  the  Complements. 

The  plurality  of  the  complements  contained  in  a  serum  has  been 
proved  by  the  most  varied  experiments.  A  separation  of  the  indi- 
vidual complements  of  the  serum  has  been  undertaken  in  various 
sera  by  means  of  chemical  or  thermic  influences,3  by  binding  with 

1  Buchner,  Berl.  klin.  Wochenschr.  1901,  No.  33. 

2  Baumgarten,  ibid.,  No.  50. 

3Ehrlich  and  Morgenroth,  see  pages  11  et  seq.;  Ehrlich  and  Sachs,  pages 
195  et  seq.;  Wendelstadt,  Centralblatt  f.  Bact.  1902,  Vol.  31,  No.  11. 


COMPLEMENTIBILITY  OF  THE  AMBOCEPTORS. 


237 


blood-cells  loaded  with  amboceptors,1  by  filtration  through  porous 
filters,2  and  by  the  action  of  a  partial  anticomplement.3  But  it 
does  not  in  all  cases  require  even  these  methods  of  separation;  all 
that  is  necessary  is  a  thorough  and  continued  study  of  the  constituents 
of  the  native  serum  of  a  given  species.  Variations  can  thus  be  observed 
therein  which  lead  at  once  to  the  view  of  a  plurality  of  complements. 

After  several  years'  observation  we  found  horse  serum  to  be 
of  especial  interest  in  this  respect,  and  we  shall  therefore  briefly 
discuss  the  complements  of  this  serum. 

Horse  serum  is  particularly  well  adapted  for  complementing 
experiments,  because,  as  a  rule,  it  exerts  but  slight  haemolytic  effect 
by  itself.  Sheep  blood,  ox  blood,  goose  blood,  and  others,  so  far 
as  we  know,  are  not  dissolved  at  all  by  horse  serum,  while  so  far  as 
guinea-pig  blood  and  rabbit  blood  are  concerned  there  is  an  extraor- 
dinary amount  of  variation,  some  horse  sera  exerting  considerable 
hsemolytic  effect  on  one  or  both  of  these  blood  species,  others  having 
no  effect  whatsoever.  In  this  respect  not*only  did  the  sera  of  different 
horses  behave  quite  differently,  but  we  also  observed  marked  chrono- 
logical variations  in  the  serum  of  one  and  the  same  normal  horse. 
These  show  how  much  the  ha^molytic  properties  of  an  individual's 
serum  can  vary.  The  behavior  of  the  serum  (always  examined  in  the 
fresh  condition)  on  the  different  days  is  seen  in  the  following  table: 

TABLE  III. 


Haemolysis  of 

Date. 

Amount  of 
Serum. 

Rabbit  Blood 
(5%  1.0). 

Guinea-pig  Blood 
(5%  1.0). 

June  19   

2  0 

very  little 

o 

1.5 

trace 

0 

0.5 

0 

0 

June  22  

2.0 
1.5 

trace 
minimal 

complete 

1.0 

1  1 

little 

0.5 

0 

1  1 

July  15  

2  0 

complete 

o 

0.6 

(  t 

0 

0.3 

strong 

0 

1  Ehrlich  and  Sachs,  1.  c. 

2  Ehrlich  and  Morgenroth,   page   56;    E.   Neisser  and  Doring,   Berl.   klin. 
Wochenschr.  1901,  No.  22. 

3  Marshall  and  Morgenroth,  pages  222  et  seq. 


238 


COLLECTED  STUDIES  IN  IMMUNITY. 


Hence  within  three  days  the  serum  of  the  horse  has  become 
strongly  hsemolytic  for  guinea-pig  blood  without  altering  its  haemo- 
lytic  property  for  rabbit  blood,  whereas  within  a  further  three  weeks 
its  properties  have  almost  become  reversed,  since  now  it  does  not 
dissolve  guinea-pig  blood  at  all,  and  dissolves  rabbit  blood  (which 
at  first  was  but  slightly  affected)  very  strongly.  It  is  worthy  of 
note  that  in  almost  every  horse  serum  which  we  examined  for  the 
purpose  we  found  a  considerable  amount  of  amboceptor  for  guinea- 
pig  blood.  This  amboceptor  was  characterized  by  a  particularly 
high  degree  of  thermolability,  being  invariably  destroyed  by  heat- 
ing to  55°  C.  A  complement  for  the  same  is  very  often  absent,  and 
even  when  present  it  is  only  on  the  addition  of  considerable  amounts 
of  fresh  guinea-pig  serum  that  this  amboceptor  becomes  manifest. 

The  cause  of  this  varying  hsemolytic  property  of  the  horse  serum, 
which  is  in  contrast  to  the  extraordinarily  constant  amount  of  normal 
tuemolysin  present  in  other  sera,  e.g.  goat  serum  and  dog  serum,  is 
perhaps  due  in  part  to  the  unusual  lability  of  the  complements  here 
concerned.  We  often  observed  that  a  horse  serum  which  dissolved 
guinea-pig  or  rabbit  blood  completely  lost  this  property,  or  nearly 
so,  by  keeping  the  serum  on  ice  for  twenty-four  hours,  a  behavior 
which  we  never  met  with  in  other  sera. 

In  a  similar  manner  horse  serum  shows  its  variability  when  it  is 
employed  purely  as  a  source  of  complement.'  We  have  frequently 
used  horse  serum  as  complement  in  the  following  combinations: 


Number. 

Blood. 

Amboceptor. 

1 

guinea-pig 

goat  serum 

2 

rabbit 

dog  serum 

3 

1  1 

ox  serum 

4 

guinea-pig 

goat  serum 

5 

dog  serum 

6 

(  i 

ox  serum 

7 

sheep 

dog  serum 

8 

serum  of  a  goat  immunized 
with  sheep  blood 

Of  all  these  cases  only  the  complement  for  6  and  for  8  was  present 
in  considerable  amounts.  So  far  as  the  other  six  complements  were 
concerned  we  observed  a  fundamental  difference  between  the  ex- 
periments which  we  had  made  some  years  ago  in  Steglitz  and  those 
made  during  the  past  two  years  in  Frankfurt.  Whereas  formerly 


COMPLEMENTIBILITY  OF  THE  AMBOCEPTORS.  239^ 

all  of  the  completions  of  normal  amboceptors  succeeded,  we  found 
in  Frankfurt  that  we  obtained  negative  results  in  the  great  majority 
of  the  experiments.  The  complements  necessary  for  the  completion 
of  almost  all  normal  amboceptors  were  absent,  while  complements 
were  present  for  a  certain  normal  amboceptor  (guinea-pig  blood,  ox 
serum),  and  for  one  obtained  by  immunizing  a  goat  with  sheep  blood.1 

This  behavior  indicates  clearly  enough  a  plurality  of  the  comple- 
ments in  a  serum,  and  we  do  not  doubt  that  further  investigations 
will  show  the  same  to  -be  true  for  the  partial  complements  of  other 
sera.  The  occasional  absence  of  one  or  the  other  complement  will 
most  easily  be  discovered  just  in  the  completion  of  normal  amboceptors, 
for  here  but  few  amboceptors  have  to  be  considered.  Of  the  numerous 
amboceptors  produced  by  immunization  in  many  cases,  at  least  a  few 
will  find  fitting  dominant  complements.  According  to  our  observa- 
tions, conclusions  can  be  drawn  only  with  the  greatest  care  from 
isolated  negative  completion  experiments.  One  cannot  conclude  that 
an  amboceptor  is  absent  from  the  impossibility  to  reactivate  normal 
inactive  sera  by  means  of  several  other  active  sera. 

For  the  evaluation  of  bactericidal  sera  in  animal  experiments 
we  believe  it  to  be  especially  important  to  consider  cases  of  this 
kind.  The  entire  absence  or  a  marked  diminution  of  complements  2 
which  functionate  as  dominant  complements  for  certain  bactericidal 
amboceptors  may  lead  to  a  disturbance  in  the  regularity  of  a  series 
of  experiments,  disturbances  which  show  themselves  in  the  fact  that 
now  and  then  an  animal  dies  of  the  infection  even  though  in  the  zone 
of  sufficient  immune  serum  to  protect  the  animal.  Such  irregularities 
are  quite  common  in  the  usual  test  series  and  manifest  themselves 
frequently  in  the  evaluation  of  bactericidal  sera,  where  they  then  are 
very  disturbing. 

1  In  respect  to  its  complements  horse  serum  occupies  a  special  place  among 
most  other  sera  used  in  the  laboratory.     Thus,  for  example,  we  were  rarely 
successful  in  complementing  the  amboceptor  of  a  rabbit  immunized  with  ox 
blood;  we  never  found  a  complement  in  horse  sera  for  the  amboceptors  of  geese 
or  goats  immunized  with  ox  blood.     That  the  locality  plays  a  certain  role  in 
these  phenomena  follows  from  our  observations  that  here,  in  contrast  to  the 
statements  of  so  reliable  an  observer  as  P.  Miiller  in  Graz,  rabbit  blood  is  not 
dissolved  by  duck  serum  to  any  appreciable  extent. 

2  Another  abnormal  phenomenon  which  is  often  observed  in  this  connec- 
tion, the  disturbing  action  of  large  amounts  of  the  immune  serum,  is  explained 
by  the   peculiar   deflection  of  complements  by  an  excess  of  amboceptor,   as 
has  been  determined  by  M.  Neisser  and  Wechsberg  (see  pages  120  et  seq .). 


240  COLLECTED  STUDIES  IN  IMMUNITY. 

It  is  hardly  to  be  doubted  that  such  variations  of  the  complement 
are  responsible  for  the  occasional  failures  of  bactericidal  sera  in 
practice,  especially  if  we  consider  that  in  diseased  conditions  a  marked 
diminution  or  a  disappearance  of  the  complements  can  take  place 
(Ehrlich  and  Morgenroth,  Metchnikoff,  Wassermann,  Schiitze  and 
Scheller). 


XXIII.     THE  PRODUCTION  OF  ILEMOLYTIC  AMBOCEP- 
TORS  BY  MEANS  OF  SERUM  INJECTIONS.1 

A  Contribution  to  Our  Knowledge  of  Receptors. 

By  J.  MORGENROTH,  Member  of  the  Institute. 

As  a  result  of  the  side-chain  theory  of  immunity,  and  especially 
in  consequence  of  the  conception  of  "receptor"  which  this  theory 
brings  with  it,  our  views  concerning  the  cytotoxins  have  to  a  great 
extent  been  emancipated  from  the  morphological  point  of  view  and 
placed  on  a  chemical  basis.  This  is  seen  most  clearly  by  looking  at 
the  complex  hsemolysins  of  serum,  for  of  all  the  various  cytotoxins 
these  have  been  most  clearly  analyzed. 

As  is  well  known,  if  an  animal  is  injected  with  erythrocytes  of  a 
foreign  species,  there  develop  in  the  serum  of  this  animal  new  sub- 
stances, the  hcemolytic  amboceptors  (immune  bodies).  The  ambo- 
ceptors  are  bound,  above  all,  by  the  red  blood-cells  of  that  species 
whose  blood  was  used  for  the  injection,  and  it  is  through  this  binding 
that  the  amboceptors  make  possible  the  haBmolytic  action  of  the 
complement  contained  in  fresh  serum.  According  to  the  side-chain 
theory  the  anchoring  of  the  amboceptors  is  the  result  of  chemical 
processes,  which  again  are  based  on  the  existence  of  certain  groups 
of  the  blood-cells'  protoplasm,  the  receptors.  If  on  the  basis  of  this 
theory  one  has  once  clearly  seen  that  the  specific  binding  is  strictly 
a  chemical  reaction  between  receptor  and  amboceptor  (or  rather 
between  their  haptophore  groups),  it  becomes  quite  evident  that  the 
morphological  structure  of  the  cell  concerned  in  the  reaction  is  some- 
thing quite  secondary.  This  is,  of  course,  apart  from  certain  prac- 
tical points  which  are  mainly  the  indicators  of  the  deleterious  action 
exerted  by  the  coaction  of  amboceptor  and  complement.  Among 
these  would  be,  in  this  case,  escape  of  haemoglobin;  in  the  cases  of 
other  cytotoxins,  disintegration  and  solution  of  the  cell,  cessation 

1  Reprint  from  the  Munch,  med.  Wochenschr.  1902,  No.  25. 

241 


242  COLLECTED  STUDIES  IN  IMMUNITY. 

of  the  motion  of  flagella  and  cilia.  The  specific  binding  of  the  am- 
boceptors  is  therefore  not  dependent  on  a  coarser  or  finer  morpho- 
logical structure:  it  can  occur  wherever  the  specifically  related  receptors 
are  present. 

For  the  doctrine  of  immunity  these  views  constitute  a  new  and 
really  concise  definition  of  specificity.  The  latter  thus  loses  the 
systematic  character  originally  given  it  by  botany  and  zoology  and 
must  from  now  on  be  regarded  purely  chemically,  as  absolutely 
dependent  on  the  conceptions  as  to  the  nature  of  the  cell's  receptors. 
Every  product  of  immunization  is  specific  for  those  receptors  by  which 
it  was  called  forth,  irrespective  of  where  the  receptors  may  be.1  When 
injected  into  an  animal  the  receptor  produces  antibodies,  and  these 
again,  when  they  meet  the  receptor  under  suitable  conditions,  are 
bound  by  the  receptor.  This  binding,  in  our  conception,  always 
remains  specific.  It  matters  not  whether  the  receptor  is  peculiar 
to  the  protoplasm  of  that  species  of  cell  which  originally  excited  the 
immunity,  or  whether  it  belongs  to  a  different  kind  of  cell  of  the 
same  species  or  to  one  of  a  strange  species. 

Hence  the  principle  of  specificity  of  the  amboceptors  produced  by  immu- 
nization is  not  violated  when,  for  example,  v.  Dungern  obtains  hsemolytic 
amboceptors  by  injections  of  ciliated  epithelial  debris,  such  as  is  contained  in 
goat  milk.  v.  Dungern  2  has  very  properly  pointed  out  this  fact  in  emphasizing 
the  community  of  the  receptors.  The  same  holds  true  for  the  haemolytic  am- 
boceptors obtained  by  Moxter  3  by  injections  of  spermatozoa.  Several  different 
zoological  species,  such  as  goat,  sheep,  and  ox,  possess  a  number  of  common 
receptors  in  their  blood-cells.4 

On  the  basis  of  the  side-chain  theory  as  it  has  just  been  laid 
down  it  is  almost  a  matter  of  course  that  these  receptors  of  the 
protoplasm  which  excite  the  production  of  the  amboceptors  are 
normally  present  dissolved  in  the  body  fluids,  a  physiological  proto- 
type of  what  occurs  to  such  a  high  degree  in  consequence  of  immu- 
nization.5 

1  See  the  explanations  by  Ehrlich  concerning  the  receptor  apparatus  of  the 
red  blood-cells  in  Schlussbetrachtungen,  Vol.  VIII,  of  Nothnagels  spezielle 
Pathol.  und  Therapie,  Vienna,  1901. 

2 -y.  Dungern,  Munch,  med.  Wochenschr.  1899,  No.  38. 

3  Moxter,  Deutsche  med.  Wochenschr.  1900,  No.  1. 

4  Ehrlich  and  Morgenroth,  page  88. 

5  It  has  already  been  shown  that  as  a  result  of  injection  of  amboceptors  into 
sensitive  animals  a  considerable  number  of  cell  receptors  are  thrust  off,  which 


PRODUCTION  OF  HJEMOLYTIC  AMBOCEPTORS.  243 

The  extraordinary  multiplicity  of  such  dissolved  substances  in 
blood  serum  has  already  been  pointed  out  by  Ehrlich.1  "The  chief 
tools  of  the  internal  metabolism  are  the  receptors  of  the  first,  second, 
and  third  order.  They  are  constantly  being  used  up  and  produced 
anew,  and  can  readily  therefore,  when  overproduced,  get  into  the 
circulation.  Considering  the  large  number  of  organs  and  the  com- 
plexity of  the  protoplasm's  chemistry  it  need  not  be  surprising  if 
the  blood,  the  representative  of  all  the  tissues,  is  filled  with  an  infinite 
number  of  the  most  diverse  receptors.  Of  these  we  have  thus  far 
learned  to  distinguish  the  various  kinds  of  lysins,  agglutinins,  coagu- 
lins,  complements,  ferments,  antitoxins,  anticomplements,  and  anti- 
ferments." 

These  free  receptors  when  injected  into  a  suitable  foreign  animal 
species  should  therefore  show  their  identity  with  those  of  the  cells 
by  the  fact  that,  like  the  latter,  they  produce  immune  bodies  identical 
with  those  produced  in  the  usual  way. 

A  few  isolated  observations  have  been  made  in  this  direction,, 
but  the  conclusions  following  therefrom  according  to  the  theory  have 
not  been  drawn.  Thus  v.  Dungern2  has  observed  the  development 
of  a  haemolysin  directed  against  chicken  erythrocytes  as  a  result  of 
injections  of  chicken  serum  into  guinea-pig  serum,  and  Tschistovitsch  3 
has  observed  the  formation  of  a  haemolysin  (besides  agglutinins)  on 
injecting  rabbits  with  horse  serum.4 

For  some  time  past  I  have  made  experiments  of  this  kind  to  demon- 
strate the  existence  hi  goat  serum  of  free  receptors  identical  with 
receptors  of  goat  erythrocytes.  These  studies  were  prompted  by 
the  observation  that  a  few  normal  goat  sera  exerted  a  slight  inhibiting 
action  on  the  amboceptors  of  rabbits  immunized  with  ox  blood,  an 
action  which  Ehrlich  and  Morgenroth  had  shown  to  be  due  to  an 
anti-immune  body.5  I  am  led  to  publish  these  experiments  now 


then  functionate  as  anti-immune  bodies.     See  Ehrlich  and  Morgenroth,  pages 
23  and  88. 

1  Ehrlich,  Schlussbetrachtungen,  1.  c. 

2  v.  Dungern,  Munch,  med.  Wochenschr.  1899. 

3  Tschistovitsch,  Annal.  Inst.  Pasteur,  1899. 

4  The  increase  in  haemolytic  action  of  rabbit  serum  for  chicken  blood  after 
the  injection  of  chicken  blood-plasma,  described  by  Xolf  (Annal.  Inst.  Pasteur, 
1901),  rests  apparently  only  on  an  increase  of  complement,  not  on  the  develop- 
ment of  new  amboceptors. 

6  See  pages  88  et  seq. 


244  COLLECTED  STUDIES  IN  IMMUNITY. 

because  of  a  rather  important  contradiction  which  exists  between 
them  and  certain  experiments  recently  published  by  Schattenfroh.1 
This  author  found  that  one  can  produce  hocmolytic  immune  bodies  for 
goat  blood  by  injecting  rabbits  with  goat  urine.  He  was  unable, 
however,  to  obtain  these  immune  bodies  by  injection  of  the  corre- 
sponding serum.  It  must  at  once  be  regarded  as  extraordinary  that 
immune  bodies  which  evidently  are  excreted  through  the  kidney  regu- 
larly and  plentifully  should  be  absent  from  the  serum  itself.  It 
would,  of  course,  have  been  possible  to  say  that  the  concentration 
of  the  receptors  in  the  serum  was  small  compared  to  that  in  the  urine, 
as  is  the  case,  for  example,  with  urea,  uric  acid,  and  other  substances. 
But  the  casual  antiamboceptor  action  of  the  serum  prevented  this, 
and  pointed  to  the  presence  in  this  of  the  dissolved  receptors.  As  a 
matter  of  fact,  therefore,  the  " interesting  contradiction"  described 
by  Schattenfroh  as  existing  between  the  action  of  the  urine  and  the 
serum  does  not  obtain;  for  it  is  possible  by  injecting  rabbits  with 
goat  serum  completely  deprived  of  blood-cells  to  produce  specific 
amboceptors.  These  amboceptors,  to  be  sure,  do  not  become  mani- 
fest if  the  usual  methods  of  investigation,  such  as  have  been  em- 
ployed by  Schattenfroh,  are  followed.  They  are,  however,  readily  and 
surely  demonstrated  if  one  attends  to  certain  fine  details. 

As  a  rule  a  hsemolytic  serum  obtained  by  specific  immunization 
will,  when  fresh,  dissolve  the  corresponding  blood-cells;  for,  as  v. 
Dungern  has  shown,  in  immunization  with  blood-cells  the  comple- 
ments usually  do  not  in  any  sense  suffer  a  change.  Only  one  excep- 
tion is  thus  far  known  in  this  respect,  namely,  the  injection  of  goat 
serum  into  the  organism  of  a  rabbit.  Ehrlich  and  Moregnroth  2  have 
shown  that  the  injection  of  goat  serum  into  rabbits  is  followed  by 
the  loss  of  certain  complements  of  the  rabbit  serum,  a  loss  which  is 
caused  by  the  development  of  anticomplements  directed  against 
the  complements  of  their  own  serum.  These  anticomplements  are 
therefore  to  be  regarded  as  auto-anticomplements.  They  not  only 
suffice  to  neutralize  the  complements  present  in  the  serum,  but  are 
able  to  bind  complement  subsequently  added.  Thus  the  amboceptor 
of  a  rabbit  mixed  with  goat  serum  is  completely  obscured;  for  if 
the  immune  serum  is  employed  fresh,  the  fitting  complements  enabling 
it  to  act  are  lacking,  while  if  the  serum  is  inactivated  and  one  seeks 


1  Munch,  med.  Wochenschr.  1901,  No.  31. 

2  See  pages  71  et  seq. 


PRODUCTION   OF  H^MOLYTIC  AMBOCEPTORS.  245 

to  activate  it  by  the  addition  of  normal  rabbit  serum,  the  comple- 
ments of  the  latter  will  be  made  inert  by  the  auto-anticomplement 
present.  Since  these  auto-anticomplements,  however,  have  no  in- 
fluence on  the  binding  of  the  amboceptor,  the  rational  mode  of  pro- 
cedure is  at  once  indicated.  The  blood-cells  are  mixed  with  the  serum 
of  the  immunized  rabbits  and  the  mixture  allowed  to  stand  until 
the  amboceptors  present  have  been  bound  by  the  blood-cells.  rihe 
latter  are  then  separated  by  centrifuge,  the  supernatant  fluid  which 
contains  the  cause  of  the  trouble,  the  auto-anticomplement,  being 
removed.  If  the  blood-cells  are  now  mixed  with  fresh  normal  rabbit 
serum,  the  haemolysis  which  ensues  in  the  incubator  will  show  the 
presence  of  the  anchored  amboceptor.  Should  this  method,  which 
guards  against  all  errors,  prove  successful,  one  can  also  get  round 
the  difficulty  in  an  easier  manner  by  using  guinea-pig  serum  as  com- 
plement. Against  this  serum,  according  to  our  experience,  the  auto- 
anticomplement  is  ineffective.  This  method,  however,  does  not 
suffice  if  we  wish  to  obtain  results  which  permit  of  only  one  inter- 
pretation. In  order  surely  to  avoid  another  source  of  error  it  is 
well  to  modify  the  test  still  further. 

It  has  been  found  that  normal  rabbit  serum  possesses  a  con- 
siderable though  variable  haemolytic  action  for  goat  blood  (see 
Table  I).  The  question  whether  we  are  dealing  with  an  amboceptor 
artificially  produced  or  with  one  which  was  originally  present  requires 
detailed  preliminary  examination  and  control  tests,  and  even  then  is 
very  uncertain  because  the  amboceptor  normally  present  finds  a 
supply  of  complement  in  guinea-pig  serum  more  plentiful  even  than 
that  in  rabbit  serum  itself,  as  can  be  seen  on  reference  to  the  table. 
This  difficulty  is  avoided  without  further  trouble  if  the  amboceptors 
produced  by  immunization  and  which  it  is  desired  to  find  are  taken 
out  of  the  fluid  by  means  of  ox  blood-cells  instead  of  goat  blood-cells. 
Because  of  the  partial  community  of  receptor  of  these  two  blood- 
cells  this  is  perfectly  allowable.  As  a  rule,  too,  normal  rabbit  serum 
dissolves  ox  blood  only  very  little,  even  though  considerable  comple- 
ment is  present.  (See  Table  I.) 

The  experiments  from  which  the  conclusions  are  drawn  in  this 
study  were  therefore  always  made  with  ox  blood.  One  cc.  of  a  5^ 
suspension  of  ox  blood-cells  is  mixed  with  varying  amounts  of  serum 
from  a  rabbit  immunized  with  goat  serum,  the  mixture  kept  at  38°  C. 
on  a  water-bath  for  one  hour,  then  centrifuged,  and  either  fresh  rabbit 
serum  added  after  the  supernatant  fluid  had  been  decanted,  or  acti- 


246 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE  I. 

HAEMOLYSIS  OF  GOAT  BLOOD  (1  CC.  5%)  BY  FRESH    SERUM  OF  NORMAL   RABBITS. 


Rabbit 
Serum. 

I. 

II. 

III. 

IV. 

V. 

VI. 

VII. 

0.25 
0.1 
0.05 

strong 
moderate 
very  little 

moderate 
little 
trace 

little 
0 
0 

moderate 
very  little 
0 

complete 
very  little 

little 
0 
0 

fair 
0 
0 

HAEMOLYSIS  OF  GOAT  BLOOD  BY  THE  SAME  RABBIT  SERA  ACTIVATED  WITH 
0.15  GUINEA-PIG  SERUM. 


0.25 

complete 

complete 

complete 

complete 

complete 

complete 

complete 

0.1 

«« 

«• 

strong  -j 

almost 
complete 

> 

r 

strong 

'« 

0.075 

" 

" 

— 

strong 

— 

strong 

0.05 

"    \ 

almost 
complete 

*    - 

— 

" 

— 

— 

0.025 

~ 

HAEMOLYSIS  OF  Ox  BLOOD  BY  THE  SAME  RABBIT  SERA  ACTIVATED  WITH 
0.15  GUINEA-PIG  SERUM. 


0.5 

trace 

faint  trace 

faint  trace 

faint  trace 

trace 

very  little 

fair 

0.25 

0 

0 

0 

0 

faint  trace 

trace 

moderate 

0.1 

0 

0 

0 

0 

0 

0 

little 

The  fresh  rabbit  sera,  even  in  amounts  of  0.5,  do  not  by  themselves  exert  any  haemolytic 
effect  on  ox  blood. 

vation  was  effected  by  the  addition  of  normal  guinea-pig  serum. 
The  hsemolytic  action  of  the  immune  sera  is  seen  in  Table  II. 

Rabbits  were  treated  with  goat  serum  which  had  been  carefully  freed  from 
all  blood-cells  by  continued  centrifuging.  Usually  the  serum  was  inactivated 
by  heating  it  to  55°  C.  for  half  an  hour,  then  it  was  injected  intraperitoneally. 
As  a  rule  the  animals  received  two  to  three  injections  of  increasing  doses  of  serum, 
in  all  about  35-90  cc.  More  frequent  injections  caused  no  greater  formation 
of  amboceptors,  a  behavior  which  corresponds  to  that  seen  with  the  injection 
of  ox  blood  or  goat  blood. 

These  experiments  show  that  specific  amboceptors  were  developed 
in  all  the  rabbits  treated  with  goat  serum.  Quantitatively  this  was 
subject  to  individual  fluctuations  just  as  is  seen  following  the  injec- 
tion of  blood-cells;  in  some  cases  the  development  was  quite  con- 
siderable. Most  of  the  sera  were  examined  fresh  for  their  action  on 
ox  blood,  and  invariably  showed  themselves  without  action  even  in 
doses  of  0.5  cc.1  The  addition  of  large  amounts  of  normal  rabbit 

1  The  method  here  employed  to  disclose  amboceptors  whose  presence  is 
masked  can  often  be  used  with  success.  Dr.  Marshall  and  I  shall  shortly  report 
an  analogous  case  dealing  with  the  amboceptors  of  a  pathological  exudate. 


PRODUCTION   OF  H.EMOLYTIC  AMBOCEPTORS. 


247 


TABLE  II. 
1.0  cc.  5%  Ox  BLOOD. 

A.  Blood  +  amboceptor  are  kept  at  37°  C.  for  one  hour.  After  centrifuging 
the  fluid  is  decanted  and  the  sediment  mixed  with  2  cc.  physiological  salt 
solution  and  0.2  cc.  rabbit  serum  as  complement. 

Complete  Haemolysis. 

Serum  rabbit         I 0 .05  cc. 

II 0.05  " 

"          "         III 0.25  " 

B.  BLOOD+ AMBOCEPTOR +0.1- 0.2  GUINEA-PIG  SERUM  AS  COMPLEMENT. 

Serum  rabbit     IV 0.1      cc. 

"          "  V 0.05     " 

"          "         VI 0.05     " 

"          "        VII 0.028'* 

"      VIII 0.013  " 

"          "         IX more  than  0.25     " 

X 0.05     " 

"          "         XI less  than  0.05     " 

serum  does  not  suffice  to  overcompensate  the  auto-anticomplement 
present.  For  example,  the  serum  of  rabbit  III  shows  the  following 
solvent  action  after  the  addition  of  0.6  cc.  rabbit  serum: 

0.5    cc 0  0.075cc very  little 

0.25  " trace  0.05    " "       " 

0.15  " "  0.025  " trace 

0.1    " very  little 

The  abnormal  course  of  this  slight  haemolysis  shows  very  well 
the  interference  of  anticomplement  on  the  one  hand  and  of  the 
amboceptor  on  the  other. 

The  similarity  of  the  amboceptor  produced  by  injections  of  goat 
serum  to  that  produced  by  injections  of  blood  is  more  plainly  seen 
by  the  fact  that  the  anti-immune  body  ob tamed  by  immunization 
acts  against  the  former  amboceptor  just  as  well  as  against  the  latter. 
Table  III  shows  this  behavior  very  well. 

The  anti-immune  body  used  was  contained  in  the  inactivated 
serum  of  a  goat  which  had  been  injected  several  times  with  the  serum 
of  rabbits  immunized  with  ox  blood.  0.3  cc.  of  this  anti-ftnmune 
body  serum  were  mixed  with  varying  amounts  of  the  amboceptor  sera 
to  be  tested  and  the  mixtures  kept  at  room  temperature  for  one  hour. 
Thereupon  1  cc.  of  a  5%  suspension  of  ox  blood-cells  was  added  to 


COLLECTED  STUDIES  IN  IMMUNITY. 


each  specimen,  which  was  then  kept  on  a  water-bath  at  38°  C.  for 
one  hour,  after  which  the  mixtures  were  centrifuged.  The  blood- 
cell  sediment  was  again  suspended  in  salt  solution  and  0.15  cc.  guinea- 
pig  serum  added  as  complement.  The  solution  which  then  ensued 
was  a  measure  for  the  bound  amboceptor,  or  for  the  deflection  by  the 
antiamboceptor.  Control  tests  were  made  with  0.3  cc.  normal  in- 
active goat  serum  in  parallel  experiments. 

TABLE  III. 

A.  INHIBITION  OF  THE  AMBOCEPTOR  OF  THE  RABBIT 
TREATED  WITH  GOAT  SERUM. 


Amount  of 
Amboceptor. 

+  0.3  Antiamboceptor. 

+  0.3  Normal  Inactive 
Goat  Serum. 

0.25 

complete  solution 

complete  solution 

0.15 

strong 

0.1 

little 

((              n 

0.075 

very  little 

(i              « 

0.05 

0 

«              (t 

0.025 

0 

strong 

B.  INHIBITION  OF  THE  AMBOCEPTOR  OF  THE  RABBIT 
TREATED  WITH  GOAT  BLOOD. 


0.2 

complete  solution 

complete  solution 

0.15 

strong 

(  (               i  ( 

0.1 

little 

<  {               1  1 

0.075 

trace 

t  <               {  t 

0.06 

0 

(  t               i  ( 

0.05 

0 

moderate 

0.025 

0 

little 

0.012 

0 

trace 

0.009 

0 

0 

The  antiamboceptor  is  thus  seen  to  offer  exactly  the  same  pro- 
tection against  the  amboceptors  obtained  as  a  result  of  goat-blood 
injections  and  those  resulting  from  goat-serum  injections,  whereby 
their  identity  is  demonstrated. 

The  presence  of  free  receptors  in  the  urine  and  serum  leads  to  the 
conclusion  that  an  active  receptor  metabolism  exists  in  the  organism 
of  the  goat;  in  other  words,  that  receptors  are  constantly  reaching 
the  serum  from  the  cells  and  are  then  excreted  by  the  kidney. 
Whether  one  is  here  dealing  with  decomposition  products  or  with 
the  products  of  some  secretion  or  other  cannot  be  determined.  The 


PRODUCTION  OF  H^MOLYTIC  AMBOCEPTORS.  249 

faet  that  free  receptors  leave  the  serum  to  reappear  in  the  urine  seems 
to  make  it  probable  that  they  have  no  significance  for  the  organism 
itself.  On  the  contrary,  one  may  suspect  that  these  are  products 
of  regressive  metabolism  which  are  eliminated  from  the  body  as 
useless.  The  explanation  that  the  free  receptors  originate  from  the 
breaking  down  of  red  blood-cells  or  other  cells  is  entirely  sufficient. 
It  may  be,  however,  that  there  is  a  physiological  thrusting-off  of 
the  same  which  bears  some  relation  to  their  nutritive  function.  In 
view  of  the  elimination  through  the  urine,  it  seems  improbable  that 
this  constitutes  a  regular  function  as  anti-immune  body  against  the 
action  of  a  possible  autolysin.  That  certainly  would  be  an  unsuita- 
ble process.  In  fact  the  free  receptors  evidently  do  not  generally 
possess  the  character  of  antiautolysins,  as  Besredka  1  believes,  for 
by  injecting  a  rabbit  with  ox  serum  it  was  impossible  to  obtain  any 
haemolytic  amboceptors.  This  corresponds  to  the  negative  results 
obtained  by  London  2  on  injecting  guinea-pigs  with  rabbit  serum. 

One  thing  is  clearly  shown  by  the  presence  of  dissolved  substances 
capable  of  producing  amboceptors,  namely,  that  without  the  idea 
of  "receptors"  a  universally  applicable  conception  of  the  origin  and 
mode  of  action  of  the  cy  to  toxins  is  impossible,  as  is  also  a  clear  con- 
ception of  the  nature  of  "specificity." 

Subsequent  Note. — In  a  recently  published  study  (Munch,  med.  Wochen- 
schr.  1902,  No.  32)  P.  Th.  Miiller  reports  on  the  production  of  haemolytic 
amboceptors  by  treating  pigeons  with  guinea-pig  serum,  and  he  accepts  the 
views  here  developed. 

1  Besredka,  Annal.  de  1'Institut  Pasteur,  Oct.  1901. 

2  London,  Arch,  des  Sciences  biologiques,  St.  Petersburg. 


XXIV.     THE     QUANTITATIVE     RELATIONS    BETWEEN 
AMBOCEPTOR,  COMPLEMENT,  AND  ANTICOMPLE- 

MENT.1 

By  Dr.  J.  MORGENROTH,  Member  of  the  Institute,  and  Dr.  H.  SACHS,  Assistant 

at  the  Institute. 

I.    Amounts  of  Amboceptor  and  Complement  Required. 

EVERY  laboratory  in  which  systematic  quantitative  studies  are 
made  on  haemolysis  will  have  had  encountered  the  relations  exist- 
ing in  different  cases  between  the  amounts  of  amboceptor  and  com- 
plement necessary  for  haemolysis.  Attention  was  first  called  to  these 
relations  by  v.  Dungern,2  who  described  a  hsemolytic  experiment 
with  ox  blood  +  amboceptor  from  a  rabbit  immunized  with  ox  blood 
+  rabbit  serum  as  complement.  In  this  case  he  noticed  that  in  order 
to  accurately  find  the  minimal  amount  of  a  completing  serum  neces- 
sary for  haemolysis,  it  was  necessary  to  employ  a  high  multiple  of  that 
amount  of  amboceptor  which  is  sufficient  to  effect  complete  solution 
when  a  large  excess  of  complement  is  present.  In  determining  the 
amount  of  complement  required,  v.  Dungern  therefore  employed 
sixteen  times  the  required  amount  of  amboceptor.  Gruber  also  says 
recently  that  "highly  prepared  (sensitized)  human  blood-cells/'  in 
consequence  of  their  preparatory  treatment,  are  dissolved  by  a  mini- 
mum of  active  normal  serum. 

In  the  following  we  wish  to  describe  several  interesting  observations 
made  by  us  in  the  course  of  several  years. 

We  shall  begin  by  describing  a  number  of  different  cases  in  which 
the  relations  between  the  amount  of  amboceptor  necessary  for  com- 
plete solution  and  that  of  the  completing  serum  were  studied.  In 
the  experiments  1  cc.  of  a  5%  suspension  of  the  blood-cells  is  always 
used.  Especial  emphasis  is  laid  on  the  fact  that  in  the  comparative 
tests  all  the  test-tubes  contained  the  same  volume  of  fluid. 

The  first  experiments  were  made  with  sheep  blood  +  amboceptor 
of  a  goat  immunized  with  sheep  blood  +  guinea-pig  serum  as  com- 
plement. (See  Table  I.) 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  No.  35.        2  See  page  38. 

250 


AMBOCEPTOR,  COMPLEMENT,  AND  ANTICOMPLEMENT.     251 


TABLE  I. 

1  cc.  5%  SHEEP  BLOOD + AMBOCEPTOR  OF  GOATS  TREATED  WITH  SHEEP 
BLOOD  +  GUINEA-PIG  SERUM  AS  COMPLEMENT. 


Amount  of 
Amboceptor. 

Proportion  of 
the  Amounts  of 
Amboceptor. 

Amount   of 
Complement 
Sufficient  for 
Complete 
Solution. 

Proportion  of 
the  Amounts  of 
Complement. 

I. 

0.05 

IX 

0.008 

1 

0.2 

4X 

0.0025 

1 
3.2 

0.4 

8X 

0.0014 

1 
5.6 

II. 

0.025 

IX 

0.04 

1 

0.038 

1.5X 

0.025 

1 
1.6 

0.05 

2X 

0.025 

1 
1.6 

0.075 

3X 

0.02 

1 
2 

0.1 

4X 

0.016 

1 
2.5 

0.2 

8X 

0.01 

1 
4 

0.5 

20  X 

0.004 

1 
10 

III. 

0.05 

IX 

0.1 

1 

0.1 

2X 

0.03 

1 
3.3 

0.2 

4X 

0.01 

1 

10 

0.4 

8X 

0.01 

1 
10 

IV. 

0.05 

IX 

0.08 

1 

0.1 

2X 

0.015 

1 

5.3 

0.2 

4X 

0.004 

1 
20 

252 


COLLECTED  STUDIES  IN  IMMUNITY. 


The  figures  in  Table  I  show  that  in  the  four  similar  cases  here 
examined  the  relation  between  the  amount  of  amboceptor  and  of 
the  complement  required  is  such  that  in  the  presence  of  larger  amounts 
of  amboceptor  smaller  doses  of  complement  suffice  for  complete  haemolysis. 
The  relation  is  not  exactly  the  same  in  the  separate  cases,  as  can 
readily  be  seen  from  the  figures  of  columns  2  and  4.  In  one  case  (I) 
increasing  the  amboceptor  eight  times  reduced  the  amount  of  com- 
plement required  only  to  — ,  whereas  in  another  case  (IV)  increas- 

o.b 

ing  the  amount  of  amboceptor  only  four  times  reduced  the  comple- 
ment required  to  — .     This  shows  us  at  once  that  there  is  no  definite 

ratio  between  the  two  factors.     The  causes  of  this  varying  relation 
will  be  discussed  later. 

The  phenomenon  in  question  is  much  less  marked  in  the  cases 
reproduced  in  Table  II,  in  which  the  combination  was  ox  blood  -f-  the 
amboceptor  of  specifically  immunized  rabbits + guinea-pig  serum  or 
rabbit  serum  as  complement. 


TABLE  II. 

A.  1  cc.  5%  Ox  BLOOD  +  AMBOCEPTOR  OF  RABBITS  TREATED  WITH  Ox  BLOOD  + 

GUINEA-PIG  SERUM  AS  COMPLEMENT. 


Amount   of 
Amboceptor. 

Proportion  of 
the  Amounts  of 
Amboceptor. 

Amount   of 
Complement 
Sufficient  for 
Complete 
Solution. 

Proportion  of 
the  Amounts  of 
Complement. 

0.002 

IX 

0.035 

1 

0.005 

2*X 

0.015 

1 

2.3 

0.01 

5X 

0.01 

1 
3.5 

0.05 

25  X 

0.008 

1 
4.4 

0.1 

SOX 

0.008 

1 
4.4 

0.2 

100  X 

0.008 

1 
4.4 

0.4 

400  X 

0.01 

1 
3.5 

AMBOCEPTOR,  COMPLEMENT,  AND  ANT1COMPLEMENT.       253 


TABLE   II — Continued. 
B.  THE  SAME,  BUT  RABBIT  SERUM  AS  COMPLEMENT. 


Amount  of 
Amboceptor. 

Proportion  of 
the  Amounts  oi 
Amboceptor. 

Amount  of 
Complement 
Sufficient  for 
Complete 
Solution. 

Proportion  ol 
the  Amounts  o 
Complement. 

I. 

0.005 

IX 

0.5 

1 

0.01 

2X 

0.17 

1 
2.9 

0.05 

10  X 

0.12 

1 
4.2 

0.1 

20  X 

0.14 

1 
3.6 

0.2 

40  X 

0.14 

1 
3.6 

0.4 

SOX 

0.15 

1 
3.3 

II. 

0.005 

IX 

0.6 

1 

0.01 

2X 

0.17 

1 
2.5 

0.05 

10  X 

0.12 

1 
5 

0.1 

20  X 

0.14 

1 
4.3 

0.2 

40X 

0.14 

1 
4.3 

0.4 

SOX 

0.15 

1 
4 

III. 

0.005 

IX 

0.75 

1 

0.0075 

1JX 

0.6 

1 
1.25 

0.015 

3X 

0.14 

1 
5.3 

0.03 

6X 

0.17 

1 
4.4 

0.06 

12  X 

0.14 

1 
5.3 

0.12 

24  X 

0.12 

1 
6.3 

254  COLLECTED  STUDIES  IN  IMMUNITY. 

Here  we  see  that  the  employment  even  of  very  high  multiples  of  the 
amboceptor  effects  a  reduction  in  the  amount  of  complement  required 
of  one-third  to  one-sixth  at  the  most.  But  what  is  particularly  char- 
acteristic for  this  case  is  the  fact  that  the  minimal  amount  of  com- 
plement is  almost  reached  with  a  small  multiple  of  the  "amboceptor 
unit,"  1  and  that  it  does  not  materially  change  with  a  further  in- 
crease of  the  amboceptor.  Thus,  in  Table  II,  A,  we  see  that  when 
five  times  the  amboceptor  unit  is  employed  the  amount  of  comple- 
ment required  is  0.01;  when  25,  50,  or  100  times  the  unit  is  employed 
the  complement  is  0.008.  Table  II,  B,  shows  that  with  the  employ- 
ment of  two  to  three  times  the  amboceptor  unit  the  maximum  of 
complement  action  is  already  attained. 

An  entirely  analogous  behavior  is  shown  by  the  cases  in  Table  III,, 
in  which  the  same  blood  and  the  same  amboceptor  are  used  as  in 
Table  I,  but  in  which  different  kinds  of  complement  are  added,, 
namely,  sheep  serum  and  horse  serum. 

These  cases  constitute  the  transition  to  those  reproduced  in  Table 
IV  which  deal  with  ox  blood  +  the  amboceptor  of  goats  treated  with 
ox  blood  +  three  different  complements,  namely,  guinea-pig,  rabbit, 
and  sheep  serum  respectively.  In  these  also  a  limit  is  reached  beyond 
which  the  decrease  of  complement  required  is  but  slightly  or  not  at  all 
affected  by  an  increase  in  the  amount  of  amboceptor. 

We  see  therefore  that  with  an  increase  of  the  amount  of  amboceptor 
the  amount  of  complement  required  at  one  time  drops  to  a  greater  or  less 
degree,  at  another  time  it  remains  unchanged.  Upon  what  does  this 
phenomenon  depend?  In  order  to  explain  this  we  must  consider  three 
factors  which  may  be  combined  with  one  another,  and  which  must  be 
considered  in  each  individual  case.  These  are:  1.  The  receptors 
present  in  the  red  blood-cell.  2.  The  conditions  of  affinity.  3.  The 
plurality  of  the  amboceptors. 

So  far  as  the  first  point  is  concerned  we  know  that  the  amount  of 
receptors  of  the  red  blood-cells  may  exhibit  great  differences  in  any 
individual  case.2 

1  We  use  the  term  "  amboceptor  unit"  to  specify  that  amount  of  amboceptor 
which  on  the  addition  of  the  optimal  amount  of  complement  just  suffices  for  com- 
plete haemolysis.     In  the  same  sense  R.  Pfeiffer  uses  the  term  "immunity  unit" 
when  speaking  of  bactericidal  sera.     Corresponding  to  the  amboceptor  unit 
the  "receptor  unit"  is  that  amount  of  receptor  which  binds  the  amboceptor 
unit. 

2  See  Ehrlich,    Schlussbetrachtungen   in   Nothnagels   spec.    Pathologic   und 
Therapie,  Vol.  VIII,  Vienna,  Holder,  1901 ;  and  Ehrlich  and  Morgenroth,  page  71. 


AMBOCEPTOR,   COMPLEMENT,  AND  ANTICOMPLEMENT.       255 


TABLE  III. 

A.    1  cc.  5%  SHEEP  BLOOD  +  AMBOCEPTOR  OF  GOATS  TREATED  WITH  SHEEP 
BLOOD  +  SHEEP  SERUM  AS  COMPLEMENT. 

B.  THE  SAME,  BUT  WITH  HORSE  SERUM  AS  COMPLEMENT. 


Amount  of  the 
Amboceptor. 

Proportion  of 
the  Amount  of 
Amboceptor. 

Amount    of 
Complement 
which  Suffices 
for  Complete 
Solution. 

Proportion  of 
the  Amounts  of 
Complement. 

A. 

0.1 

1      X 

0.15 

1 

0.25 

2.5X 

0.035 

1 
4.3 

0.5 

5     X 

0.05 

1 
3 

0.75 

7.5X 

0.05-0.035 

ltoo 

B. 

0.  1 
0.2 

IX 
2X 

0  .  5  almost 
[complete 

1 
5 

0.4 

4X 

0.1 

1 
5 

0.8 

8X 

0.1 

1 
5 

One  erythrocyte  may  possess  just  so  many  receptors  for  a  cer- 
tain poison  as  are  necessary  to  bind  a  single  solvent  dose,  ie.  there 
is  present  just  a  receptor  unit,  whereas  in  other  cases  such  a  multiple 
of  the  receptor  unit  may  be  present  that  a  hundred  times  the  ambo- 
ceptor  unit  is  bound.  In  bacteria  the  latter  condition  is  present  to  a 
still  very  much  greater  degree:  agglutinins  (Eisenberg  and  Volk) 
and  bacteriolytic  amboceptors  (R.  Pfeiffer)  are  bound  in  enormous 
excess,  frequently  as  high  as  many  thousand  times  the  effective 
amount.  It  is  therefore  entirely  clear  that  these  conditions  must 
exercise  a  deciding  influence  on  the  fact  whether  an  increased  amount 
of  immune  serum  decreases  the  amount  of  complement  required 
or  not.  It  may  be  regarded  as  self-evident  that  in  all  those  cases 
in  which  only  the  single  effective  dose  can  be  bound,  i.e.  in  which 
only  one  amboceptor  unit  is  anchored,  an  excess  of  amboceptor  can 
never  exert  a  favorable  influence;  on  the  contrary  an  increase  in  the 


256 


COLLECTED  STUDIES  IN   IMMUNITY. 


amount  of  complement  can  readily  result  owing  to  the  deflection 
phenomenon  whose  significance  was  first  pointed  out  by  M.  Neisser 
and  Wechsberg.1 

TABLE  IV. 

A.   1  cc.  5%  Ox  BLOOD  +  AMBOCEPTOR  OF  GOATS  TREATED  WITH  Ox  BLOOD + 
GUINEA-PIG  SERUM  AS  COMPLEMENT. 

B.  THE  SAME  +  RABBIT  SERUM  AS  COMPLEMENT. 
C.  THE  SAME  +  SHEEP  SERUM  AS  COMPLEMENT. 


Amount  of  the 
Amboceptor. 

Proportion  of 
the  Amounts  of 
Amboceptor. 

Amount  of 
Complement 
which  Suffices 
for  Complete 
Solution. 

Proportion  of 
the  Amounts  ol 
Complement. 

A. 

0.1 

IX 

0.01 

1 

0.2 

2X 

0.01 

1 

0.4 

4X 

0.01 

1 

0.8 

8X 

0.01 

1 

B. 

0.1 

IX 

0.15 

1 

0.2 

2X 

0.15 

1 

0.4 

4X 

0.15 

1 

0.8 

8X 

0.15 

1 

C. 

0.1 

IX 

0.1 

1 

0.2 

2X 

0.1 

1 

0.4 

4X 

0.1 

1 

0.8 

8X 

0.075 

1 

1.4 

The  problem  is  more  difficult  in  those  cases  in  which  the  red  blood- 
cells  contain  a  plurality  of  receptor  units  arid  therefore  bind  a  mul- 
tiple of  amboceptor  units.  In  these  cases  the  result  of  the  experi- 
ments will  depend  mainly  on  the  following  factors. 

We  know  that  as  a  rule  the  affinity  of  the  amboceptor's  comple- 
mentophile  group  is  increased  when  the  cytophile  group  is  anchored 
by  the  receptors.  If  this  relative  increase  of  affinity  is  very  large, 
the  added  complement  will  combine  exclusively  with  the  anchored 
amboceptor,  and  in  certain  doses  will  effect  solution.  In  this  case 


M.  Neisser  and  Wechsberg,  see  page  120. 


AMBOCEPTOR,  COMPLEMENT,  AND  ANTICOMPLEMENT.       257 

the  required  equivalence  will  already  be  reached  with  the  amount  of 
complement  just  sufficient  for  solution,  and  an  increase  of  the  com- 
plement action  by  loading  the  blood-cells  with  additional  ambo- 
ceptor  will  not  occur. 

The  conditions,  however,  are  entirely  different  if  the  affinity  of 
the  complementophile  group  of  the  anchored  amboceptor  for  the 
complement  is  only  very  slight;  in  other  words,  when  we  are  dealing 
with  an  easily  dissociated  combination  in  a  reversible  process.  In 
that  case,  in  accordance  with  a  well-known  chemical  law,  the  more 
of  one  of  the  elements  is  in  excess,  the  more  of  the  completed  combination 
will  remain  intact.  Hence  if  there  are  very  few  receptor  units  in  the 
blood-cells,  it  will  be  necessary  to  add  very  much  complement  in  order 
to  diminish  the  amount  of  dissociation  and  to  cause  the  formation 
of  an  effective  unit  of  hsemolysin;  if  more  receptor  units  are  present, 
less  complement  will  suffice.  The  tables  here  given  present  numerous 
considerations  which  show  that  little  amboceptor + much  complement 
and  much  amboceptor  +  little  complement  lead  to  the  formation  of  the 
same  amount  of  complemznt-amboceptor  combination  (haemolysin 
unit)  anchored  by  the  receptors. 

A  most  conspicuous  role,  however,  is  played  by  the  fact  that  the 
immune  serum  is  not  a  simple  substance,  but  is  made  up  of  partial  ambo- 
ceptors  to  which  various  dominant  complements  of  the  sera  correspond. 
Of  especial  importance  in  this  respect  are  partial  amboceptors  present 
in  immune  serum  in  small  amounts  (and  which  therefore  can  only 
come  into  action  when  high  multiples  of  the  immune  serum  are 
employed) ,  but  which,  for  their  completion,  find  a  partial  complement 
which  is  particularly  plentiful  in  the  completing  serum.  Such  a 
partial  amboceptor  present  in  these  small  amounts  (such,  for  example, 
as  has  been  demonstrated  in  the  serum  of  rabbits  treated  with  ox 
blood)  constitutes  one  of  the  main  explanations  for  the  phenomena 
above  described. 

From  these  considerations  we  see  that  the  various  phenomena 
which  we  observe  in  the  interdependence  of  the  amounts  of  ambo- 
ceptor and  complement  required  may  have  entirely  different  causes, 
but  that,  by  regarding  all  of  the  three  above-mentioned  factors,  these 
phenomena  can  be  explained  very  naturally.  Under  these  circum- 
stances it  is,  of  course,  not  permissible  to  generalize  from  one  particular 
case. 


258  COLLECTED  STUDIES  IN  IMMUNITY. 


II.    Amount  of  Amboceptor  and  Anticomplement  Required. 

The  following  observations  deal  with  the  quantitative  relations 
existing  between  the  amount  of  amboceptor  and  that  of  the  anticom- 
plement  required  to  prevent  haemolysis.  In  a  number  of  cases  we 
determined  the  amount  of  anticomplement  which  just  suffices  to 
prevent  the  solution  of  red  blood-cells  loaded  with  varying  amounts 
of  amboceptor,  when  that  amount  of  complement  was  present  which 
always  just  sufficed  for  complete  solution. 

The  majority  of  our  experiments  again  refer  to  the  solution  of 
sheep  blood  by  an  immune  serum  (derived  from  a  goat)  whose  ambo- 
ceptor is  complemented  by  guinea-pig  serum.  This,  it  will  be  re- 
called, is  the  case  in  which  with  large  amounts  of  amboceptor  the 
complement  required  decreases  considerably.  For  the  anticomple- 
ment we  made  use  of  the  serum  of  a  goat  which  had  previously  been 
treated  with  repeated  injections  of  rabbit  serum.  This  serum,  as 
can  be  seen  from  a  previous  communication,  does  not  only  protect 
against  the  complement  of  rabbit  serum,  but  also  against  those  of 
guinea-pig  serum. 

To  begin,  the  amount  of  completing  guinea-pig  serum  was  deter- 
mined which,  with  varying  amounts  of  amboceptor,  sufficed  for 
the  complete  solution  of  1  cc.  5%  sheep  blood.  After  this  the  quan- 
tity of  anticomplement  required  in  each  instance  to  effect  neutrali- 
zation was  determined,  whereupon  complement  and  anticomplement 
were  mixed  and  kept  at  37°  C.  in  an  incubator  for  half  an  hour. 
Blood  and  amboceptor  were  then  added.  Such  an  experiment  is 
reproduced  in  Table  V. 

As  shown  in  the  table  by  the  degree  of  haemolysis,  the  peculiar 
behavior  is  observed  that  with  small  amounts  of  amboceptor  0.015 
cc.  anticomplement  serum  neutralize  the  complement  of  0.05  in  guinea- 
pig  serum,  whereas  with  large  amounts  of  amboceptor  0.35  cc.  anti- 
complement  serum  are  required  to  neutralize  0.006  guinea-pig  serum. 
If  we  calculate  the  amount  of  complementing  serum  neutralized  in 
both  cases  by  1  cc.  anticomplement  serum,  we  find  that  in  one  case 
it  is  3.3  cc.,  in  the  other  0.017  cc.  Hence  when  large  amounts  of  ambo- 
ceptor are  employed  the  anticomplement  acts  195  times  weaker. 
The  required  amount  of  anticomplement  is  therefore  absolutely 
dependent  on  the  quantity  of  the  amboceptor  employed.  This 
becomes  most  evident  by  the  fact  that  even  with  equal  amounts  of 


AMBOCEPTOR,  COMPLEMENT,  AND  ANTICOMPLEMENT.       259 


complement  required,  but  with  varying  additions  of  amboceptor  (see 
columns  a  and  b  of  Table  V),  different  amounts  of  anticomplement 
(corresponding  to  the  amount  of  amboceptor  present)  are  required 
to  neutralize  the  complement,  more  being  required  with  larger  amounts 
of  amboceptor.  In  these  cases,  therefore,  the  amount  of  anticomplement 
required  is  far  from  being  a  simple  function  of  the  amount  of  comple- 
ment, but  is  dependent  on  the  amount  of  amboceptor  present. 

TABLE  V. 
A. 


Amount  of  the  Amboceptor. 


Amount  of  the  Complement  Sufficient  for 
Complete  Solution. 


0.3 
0.05 
0.01 
0.005 


0.005 
0.005 
0.01 
0.035 


B. 


Amount  of 

a 

b 

c 

d 

Anticomple- 
ment. 

Amboceptor,  0.3. 
Complement,  0.006 

Amboceptor,  0.05. 
Complement,  0.006 

Amboceptor,  0.01. 
Complement,  0.01. 

Amboceptor,  0.005 
Complement,  0.05. 

0.35 

0 

0 

0 

0 

0.25 

faint  trace 

0 

0 

0 

0.15 

trace 

0 

0 

0 

0.1 

(  i 

0 

0 

0 

0.075 

moderate 

•     faint  trace 

0 

0 

0.05 

complete 

trace 

faint  trace 

0 

0.035 

ti 

moderate 

little 

0 

0.025 

<<4 

complete 

<  < 

0 

0.015 

I  f 

(  ( 

complete 

0 

0.01 

I  I 

1  1 

{  t 

faint  trace 

0 

(I 

tf 

complete 

In  several  other  combinations,  which  we  analyzed  in  a  similar 
manner,  we  met  with  the  same  behavior  to  a  greater  or  less  extent. 
In  Table  VI  such  an  experiment  is  reproduced;  it  deals  with  the 
solution  of  ox  blood  by  an  amboceptor  derived  from  rabbits  and 
complemented  by  guinea-pig  serum.  As  hi  the  previous  case,  inactive 
serum  of  a  goat  treated  with  rabbit  serum  served  as  anticomplement. 

In  this  case  when  small  amounts  of  amboceptor  are  present  1.0 
cc.  of  the  anticomplement  serum  neturalizes  1.0  cc.  guinea-pig  serum; 
with  larger  amounts  of  amboceptor  it  neutralizes  only  0.067  cc.; 
i.e.,  about  fifteen  times  less. 


260 


COLLECTED  STUDIES  IN   IMMUNITY. 


TABLE  VI. 
Ox  BLOOD +AMBOCEPTOR  OF  AN  OX-BLOOD  RABBIT  +  GUINEA-PIG  SERUM. 


Amount  of 
Amboceptor. 

Amount  of  Complement  Sufficient  to 
Effect  Complete  Solution. 

0.2 

0.05 

0.004 

0.075 

Anticomple- 

ment. 

Amboceptor,  0.2. 
Complement,  0.05. 

Amboceptor,  0.004. 
Complement,  0.1. 

0.75 

0 

0 

0.5 

strong 

0 

0.35 

almost  complete 

0 

0.25 

complete 

0 

0.15 

0 

0.1 

it 

0 

0.075 

« 

trace 

0.05 

« 

little 

0.035 

n 

moderate 

0.025 

ti 

strong 

0.015 

I  C 

almost  complete 

0.01 

{  t 

complete 

The  study  of  the  phenomena  of  immunization  has  taught  us  that 
nothing  is  so  liable  to  error  as  premature  generalization.  Hence 
we  were  not  at  all  surprised  to  find  that  there  are  cases  in  which, 
in  contrast  to  that  above  described,  the  quantity  of  anticomplement 
required  appeared  exclusively  to  be  a  function  of  the  amount  of 
complement,  and  in  no  way  dependent  on  the  degree  of  occupation 
of  the  receptors  by  amboceptors.  Curiously  enough  this  case  con- 
cerns the  combination  first  described,  namely,  sheep  blood,  ambo- 
ceptor  of  goats  treated  with  sheep  blood,  and  guinea-pig  serum  as 
complement,  with  this  difference,  however,  that  in  this  case  the  anti- 
complement  was  not  the  same,  since  it  was  derived  from  a  rabbit  treated 
with  guinea-pig  serum.  This  anticomplement,  therefore,  so  far  as  its 
relation  to  guinea-pig  serum  was  concerned,  can  be  termed  "iso- 
genic"  in  contrast  to  the  anticomplement  previously  used,  which 
can  be  termed  "alloiogenic,"  since  it  was  derived  by  injecting  rabbit 
serum.  The  experiment  is  shown  in  Table  VII. 

Here  we  see  that  neutralization  of  the  ten  times  larger  amount  of 
complement,  such  as  is  made  necessary  by  the  smaller  amount  of 
amboceptor,  requires  ten  times  as  much  anticomplement  as  it  does 
with  one-tenth  the  quantity  of  complement  when  larger  amounts 
of  amboceptor  are  used. 


AMBOCEPTOR,   COMPLEMENT,  AND  ANT1COMPLEMENT.       261 
TABLE  VII. 


Amount  of 
Amboceptor. 

Amount  of  Comple- 
ment Sufficient  for 
Complete  Solution. 

Amount  of  Comple- 
ment in  the  Anti- 
complement  Test. 

Amount  of  Anticom- 
plement  Required 
for  Complete  Neu- 
tralization 

0.1 
0.2 

0.02 
0.0025 

0.025 
0.0035 

0.04 
O.OC5 

The  results  of  the  experiments  in  the  various  cases  are  diametric- 
ally opposite,  for  in  one  case  there  is  a  relation  between  complement 
and  amount  of  anticomplement  required  with  different  quantities  of 
amboceptor,  in  other  cases  there  is  a  wide  divergence.  How  are 
these  phenomena  to  be  explained? 

To  begin,  let  us  assume  for  the  sake  of  simplicity  that  comple- 
ment and  anticomplement  are  of  simple  constitution.  In  that  case, 
if,  as  all  our  experiments  show,  the  affinity  of  complement  is  much 
greater  for  anticomplement  than  for  amboceptor,  the  neutralization 
of  complement  and  anticomplement  should  follow  stoichiometric  laws, 
As  a  matter  of  fact  this  is  wiiat  we  found  in  the  last  case  (Table  VII). 
In  the  first  two  cases,  however,  the  results  diverge  so  widely  from 
this,  and  are  moreover  so  far  beyond  the  limits  which  might  be  caused 
by  errors,  that  from  this  fact  alone  it  necessarily  follows  that  con- 
ditions of  affinity  cannot  by  themselves  suffice  for  an  explanation. 
We  are  therefore  compelled  to  call  to  our  aid  another  factor,  one 
which  we  have  already  emphasized,  namely,  the  plurality  of  the  comple- 
ments and  anticomplenients. 

Let  us  assume  that  in  this  case  two  dominant  complements, 
A  and  B,  came  into  play  in  the  complementing  serum.  The  serum 
sen- ing  as  anticomplement  must  therefore  contain  the  corresponding 
anticomplement  a  or  p.  It  is  self-evident  that  the  corresponding 
anticomplements  are  present  in  the  isogenic  serum;  that  they  may 
also  appear  in  the  serum  obtained  by  injection  of  a  different  serum, 
e.g.  of  rabbit  serum,  is  shown  by  previous  experience.  It  is  not  at 
all  necessary  to  assume  that  rabbit  serum  contains  exactly  the  same 
complements  A  and  B  present  in  guinea-pig  serum;  it  suffices  to 
assume  a  partial  identity  for  the  rabbit  serum's  complements  (Ai  and 
BI),  namely,  an  identity  in  the  haptophore  group. 

Following  the  terminology  of  the  theory  of  numbers  in  which  "friendly 
numbers"  (numeri  amicabiles)  are  spoken  of,  one  could  designate  complements 
of  different  species  which  correspond  in  their  haptophore  groups,  as  "friendly 
complements." 


262 


COLLECTED  STUDIES  IN  IMMUNITY. 


Now  if  one  injects  any  serum  containing  two  different  comple- 
ments, the  production  of  partial  anticomplements  will  to  a  great 
extent  depend  on  the  relative  amount  of  the  two  complements.  For 
example,  if  in  one  case  there  is  considerable  complement  A  and  but 
little  B,  while  in  another  case  there  is  considerable  B  and  little  A, 
the  anticomplement  will  be  directed  for  the  greater  part  against  A 
in  the  one  case,  and  against  B  in  the  other.  It  is  therefore  readily 
understood  that  with  isogenic  sera  the  yield  of  anticomplements  can 
correspond  fairly  well  to  the  mixture  of  complements  present  in  the 
injected  material,  for  the  average  composition  of  this  mixture  is 
quite  constant.  A  serum  thus  results  which  to  a  certain  extent  is 
fitted  to  the  complements  of  the  serum  injected. 

Since,  however,  a  serum  contains,  not  two  complements  as  we 
have  assumed  for  the  sake  of  simplicity,  but  a  large  number  of  com- 
plements, it  can,  of  course,  happen  even  with  isogenic  anticomple- 
ments that  a  disharmony  will  occur  so  far  as  certain  fractions  of 
complements  are  concerned.  The  following  case  shows  that  even 
with  an  isogenic  anticomplement  the  relative  proportion  between 
complement  and  anticomplement  with  different  amounts  of  ambo- 
ceptor  is  not  maintained.  (See  Table  VIII.) 

TABLE  VIII. 

HUMAN  BLOOD  +  AMBOCEPTOR  OF  A  HUMAN-BLOOD  RABBIT  +  RABBIT  SERUM -f 
ANTICOMPLEMENT  FROM  THE  GOAT  TREATED  WITH  RABBIT  SERUM. 


Amount  of  Amboceptor. 

Amount  of  Complement  Necessary  for 
Complete  Solution. 

0.2 
0.2 

0.05 

0.05 
0.05 
0.075 

Anticomplement. 

Amboceptor,  0.2. 
Complement,  0.05 

Amboceptor,  0,1. 
Complement,  0.05. 

Amboceptor,  0.05. 
Complement,  0.1. 

0.1 

0 

0 

0 

0.075 

0 

0 

0 

0.05 

trace 

0 

0 

0.035 

<  t 

0 

0 

0.025 

little 

trace 

0 

0.015 

moderate 

(  ( 

trace 

0.01 

almost  complete 

little 

moderate 

0 

complete 

complete 

complete 

In  this  case  1.0  cc.  anticomplement  neutralizes  4.0  cc.  complement  when 
0.5  cc.  amboceptors  are  present,  1.42  cc.  when  0.1  cc.  amboceptor  is  present, 
and  only  0.67  cc.  complement  with  0.2  cc.  amboceptor. 


AMBOCEPTOR,  COMPLEMENT,  AND  ANTICOMPLEMENT.       263 

A  priori,  it  is,  of  course,  conceivable  that  in  the  rabbit  the 
complements  AI  and  BI  exist  exactly  in  the  same  proportion  as  do 
complements  A  and  B  in  the  guinea-pig,  but  we  must  admit  that 
this  would  be  a  coincidence.  In  all  probability  the  development 
of  the  alloiogenic  anticomplement  will  result  hi  a  serum  hi  which  the 
proportion  of  the  two  anticomplements  is  absolutely  different,  so 
that,  for  example,  anticomplement  B  will  be  present  in  much  smaller 
amount  than  in  the  isogenic  anticomplement  serum.  The  behavior 
of  this  will  then  be  as  follows:  A  certain  quantity  of  the  isogenic 
anticomplement  serum  produced  by  guinea-pig  serum  (presupposing 
that  its  constitution  is  uniform)  will  neutralize  guinea-pig  serum  hi 
such  a  way  that  complement  A  and  complement  B  of  this  mixture 
are  neutralized  at  the  same  time.  If  we  proceed  to  do  the  same 
with  the  alloiogenic  anticomplement  serum,  we  find  that  in  the  mix- 
ture of  anticomplement  and  guinea-pig  serum,  complement  A  is 
completely  neutralized,  but  that  a  larger  or  smaller  excess  of  com- 
plement B  is  still  unsaturated.  In  those  cases  in  which  comple- 
ment A  is  the  dominant  complement  both  mixtures  will  prove  neutral; 
when  amboceptors  are  employed  for  which  B  is  the  dominant  com- 
plement, only  one  of  the  mixtures  will  be  neutral,  the  other  will  still 
be  active. 

Now  we  shall  assume  that  with  the  employment  of  large  amounts 
of  amboceptor,  a  partial  amboceptor  comes  into  action  which  is 
present  in  the  immune  serum  in  relatively  small  quantity.  This  partial 
amboceptor  is  complemented  by  complement  B  contained  in  guinea-pig 
serum,  whereas  the  preponderating  amboceptor  is  sensitized  by  comple- 
ment A.  Complement  B  finds  a  plentiful  amount  of  anticomple- 
ment in  the  isogenic  immune  serum,  but  not  hi  the  alloiogenic  serum. 
In  the  latter  case,  therefore,  disproportionately  much  serum  contain- 
ing B  anticomplement  will  be  required  in  order  to  inhibit  the  com- 
plement action  when  large  quantities  of  amboceptor  are  present.  If 
the  difference  becomes  so  great  that  the  anticomplement  against 
complement  B  is  present  only  in  very  slight  amounts,  we  shall  have 
a  condition  like  that  described  by  Marshall  and  Morgenroth  (see 
page  222).  They  found  an  ascitic  fluid  which  was  effective  only 
against  a  particular  complement  of  a  serum,  while  it  was  entirely 
inert  against  another  serum  of  this  same  species. 

We  have  endeavored  to  establish  this  point  of  view  on  a  wider 
experimental  basis.  With  this  end  in  view  we  first  used  small  amounts 
of  amboceptor,  adding  various  multiples  of  the  complementing  dose 


264 


COLLECTED   STUDIES  IN  IMMUNITY. 


of  serum  and  then  determining  the  amount  of  anticomplement 
required  in  each  case.  In  one  of  the  experiments  we  made  a  parallel 
test  with  a  large  excess  of  amboceptors.  The  results  showed  that 
under  these  circumstances,  for  each  of  the  cases  and  with  a  certain 
amount  of  amboceptor,  the  anticomplement  required  is  proportionate 
to  the  amount  of  complement.  This  is  shown  in  Table  IX. 


TABLE  IX. 

1   cc.  5%  SHEEP  BLOOD  +  AMBOCEPTOR   OF  GOATS  IMMUNIZED   WITH   SHEEP 

BLOOD  +  GUINEA-PIG  SERUM  AS  COMPLEMENT. 
The  serum  of  a  goat  treated  with  rabbit  serum,  as  anticomplement. 


Amount   of 
Amboceptor. 


Amount  of 
Complement. 


Amount  of 
Anticomplement 
Necessary  for  Com- 
plete Neutralization. 


A.  Little  Amboceptor  (  =  1  Amboceptor  Unit). 


0.005 
0.005 


0.1 
0.2 


0.22 
0.4 


B.   Much  Amboceptor  (=25  Amboceptor  Units). 


0.125 
0.125 
0.125 


0.006 
0.012 
0.024 


0.24 
0.42 
0.8 


1  cc.  5%  Ox  BLOOD + AMBOCEPTOR  OF  A  GOAT  IMMUNIZED  WITH  Ox  BLOOD + 

RABBIT  SERUM  AS  COMPLEMENT. 
The  serum  of  a  goat  treated  with  rabbit  serum  as  anticomplement. 


Amount   of 
Amboceptor. 

Amount  of 
Complement  which 
is  just  Fully 
Neutralized. 

Amount  of 
Anticomplement. 

0.15* 
0.15 
0.15 

0.2 
0.1 
0.05 

0.1 

0.05 
0.025 

*  =  about  2  amboceptor  units. 

Here,  then,  we  are  dealing  with  the  same  phenomenon  which  in 
the  domain  of  antitoxin  immunity  wre  know  as  the  multiplication  of 
the  L0  dose.  From  our  standpoint  this  is  easily  explained,  for  if 
at  any  point  in  the  saturation  of  the  blood-cells'  amboceptors  a 
certain  amount  of  the  complement  dominant  in  this  case  is  neutral- 
ized by  a  certain  quantity  of  anticomplement,  the  other  conditions 
will  in  no  way  be  altered  by  a  doubling,  quadrupling,  etc.,  of  the 


AMBOCEPTOR,   COMPLEMENT,  AND  ANTICOMPLEMENT.       265 

complement,  and  the  amount  of  complement  and  that  of  anticom- 
plement  required  remain  in  the  same  ratio.  A  definite  relation  there- 
fore exists  in  every  grade  of  amboceptor  saturation  between  the  amount 
of  complement  and  that  of  anticomplement  required.  This  is  in  con- 
trast to  the  great  differences  which  appear  when  the  occupation 
with  amboceptors  varies.  The  relation  just  described  indicates  that 
we  are  here  dealing  with  a  chemical  process  following  stoichiometric  laws. 

We  should  like  to  mention  further  that  this  peculiar  behavior  observed  by 
us  is  of  some  importance  in  refuting  an  objection  made  by  Gruber  (1.  c.)  against 
Wechsberg.  As  is  well  known,  Gruber  believed  he  had  shown  that  in  the 
bactericidal  sera  anticomplements  were  present  produced  by  the  immuniza- 
tion. This  he  held  to  be  very  important,  since  according  to  his  view  it  showed 
that  the  deflection  of  complements  by  excess  of  amboceptors,  which  had  been 
described  by  Xeisser  and  Wechsberg,  was  incorrect.  This  is  not  the  place  to 
enter  into  the  great  improbability  of  Gruber's  deductions,  for  this  has  already 
been  well  pointed  out  by  Wechsberg,  by  Lipstein,1  and  by  Levaditi.2  Wechs- 
berg 3  repeated  Gruber's  experiments,  but  was  unable  to  confirm  his  results. 
Sachs  also  was  unable  to  do  this.  Gruber  has  now  objected  to  Wechsberg's 
work  on  the  score  of  a  gross  error,  saying  that  Wechsberg  worked  with  weakly 
sensitized  blood-cells,  whereas  he  had  used  strongly  sensitized  blood-cells. 
Wechsberg  had  therefore  used  considerably  more  complement  than  he,  and 
had  in  consequence  required  much  more  anticomplement  for  neutralization, 
so  that  the  presence  of  small  quantities  of  anticomplement  could  easily  have 
escaped  Wechsberg. 

From  what  has  been  said  above,  however,  Just  the  contrary  occurs;  with  alloio- 
genic  sera  larger  amounts  of  anticomplement  are  used.  That  the  anticomple- 
ment which  would  be  produced  artificially  by  injections  of  bacteria  (even  if 
that  be  regarded  as  conceivable)  would  eminently  be  alloiogenic  need  not 
further  be  emphasized.  It  is  shown  by  Table  VIII  that  the  conditions  which 
Gruber  assumed  to  exist  do  not  obtain,  even  with  an  isogenic  anticomplement, 
in  Gruber's  case  (human  blood -f  human-blood  rabbit  +  rabbit  serum).  It  is 
unnecessary  to  enter  further  into  Gruber's  objections,  for  Wechsberg 4  has 
succeeded  through  the  demonstration  of  complementophile  amboceptoids  in 
finding  the  source  of  the  differences.  These  amboceptoids  have  meantime 
been  found  independently  by  E.  Xeisser  and  Friedemann  5  and  by  P.  Th.  Muller.8 

It  is  immaterial  in  judging  of  this  phenomenon  whether  in  the  anticomple- 
mentary  sera  used  by  Gruber  the  diverting  amboceptoids  developed  as  a  result 
of  long  standing  or  under  the  influence  of  too  high  an  inactivating  temperature. 
The  main  thing  is  that  even  the  phenomenon  observed  by  Gruber  and  used 

1  Lipstein,  see  pages  132  et  seq. 

2  Levaditi,  Compt.  rend.  Soc.  de  Biol.  1902,  No.  25. 

3  Wechsberg,  Wiener  klin.  Wochenschr.  1902,  Nos.  13  and  28. 

4  Ibid. 

*  Neisser  and  Friedemann,  Berl.  klin.  Wochenschr.  1902,  No.  29. 
8  P.  Th.  Muller,  Munch,  med.  Wochenschr.  1902,  No.  32. 


266  COLLECTED  STUDIES  IN  IMMUNITY. 

by  him  as  an  objection  constitutes  a  new  and  telling  demonstration  of  the 
correctness  of  the  amboceptor  theory. 

Thus  we  see  that  the  anticomplement  experiments  give  us  a 
further  insight  into  the  mechanism  of  hsemolysin  action.  This  in 
its  turn  shows  that  the  simple  Unitarian  conception  must  be  aban- 
doned to  be  replaced  by  the  view  maintained  by  us  that  the  exciting 
substances  as  well  as  the  reaction  products  arising  in  immunization 
are  exceedingly  manifold  in  character. 


XXV.     THE   ILEMOLYTIC  PROPERTIES  OF  ORGAN 

EXTRACTS.1 

By  Dr.  S.  KORSCHUN,  of  Charkow,  and  Dr.  J.  MORGENROTH,  Member  of  the 

Institute. 

The  first  observations  concerning  the  haemolytic  properties  of 
organ  extracts  were  published,  so  far  as  we  are  aware,  by  Metchni- 
koff.2 

Proceeding  from  his  observation  that  in  the  peritoneum  of  the 
guinea-pig  goose  blood-cells  are  taken  up  by  certain  phagocytes, 
the  macropJiages,  and  digested  intracellularly,  Metchnikoff  sought  to 
demonstrate  digestive  actions  in  vitro  in  extracts  of  such  organs 
which  are  rich  in  macrophages.  He  regarded  the  hamolytic  function 
as  an  indicator  of  this  digestive  action.  He  found  that  extracts  of 
certain  organs  of  guinea-pig  (but  not  guinea-pig  serum)  exerted  a 
ha?molytic  action  on  goose  blood;  the  lymphoid  portion  of  the  omen- 
turn  showed  this  action  quite  regularly,  the  mesenteric  glands  fre- 
quently, and  in  a  limited  number  of  cases  the  spleen.  Of  the  other 
organs  the  pancreas  showed  a  marked,  and  the  salivary  glands  a 
weak  haBmolytic  action;  the  bone  marrow,  liver,  kidney,  brain  and 
spinal  cord,  ovaries,  testicles,  and  adrenals  were  inert. 

Metchnikoff  found  the  haemolytic  substance  to  be  a  soluble  ferment 
contained  in  the  macrophages;  he  termed  it  "macrocytase"  to  dis- 
tinguish it  from  the  bactericidal  ferment  derived  from  microphages, 
which  he  calls  "microcytase."  It  shows  itself  to  be  a  "cytase"3 

1  Reprint  from  the  Berlin,  klin.  Wochenschr.  1902,  No.  37. 

2  Metchnikoff,  Annal.  de  PInstit.  Pasteur,  Oct.  1899;   see  further  references 
in  Metchnikoff,  I'lmmunite",  Paris,  1901. 

3  Metchnikoff  and  his  pupils  use  the  term  "cytase"  for  our  complements 
as  well   as   for  the  complex  cytotoxins  (hsemolysins,   bacteriolysins,  etc.)   of 
normal  sera.     It  is  to  be  regretted  that  although  in  numerous  instances  these 
have  been  shown  to  consist  of  amboceptor  and  complement  this  fact  has  not 
been  sufficiently  regarded  by  this  school  (see  especially  the  recent  studies  by 
Sachs,  pages  181  et  seq.,  and  Morgenroth  and  Sachs,  page  233). 

267 


268  COLLECTED  STUDIES  IX   IMMUNITY. 

by  its  behavior  toward  heat,  completely  losing  its  action  on  being 
heated  to  56°  C.  for  three-quarters  of  an  hour. 

Observations  in  this  same  direction  have  been  made  by  Shibayama  1 
and  Klein,2  and  a  comprehensive  study  by  Tarassevitsch  3  has  recently 
appeared  from  MetchnikofFs  laboratory. 

Shibayama,  working  in  Kitasoto's  laboratory,  studied  the  action 
of  extracts  of  guinea-pig  organs  on  dog  blood  and  obtained  haemolysis 
with  those  of  spleen  and  lymph  glands,  but  not  with  those  of  bone 
marrow  and  other  organs.  Without  further  analysis  he  classes  r.s 
identical  the  haemolytic  substances  of  the  organs  and  the  specific 
haemolysins  which  appear  in  the  serum  after  immunization  with  dog 
blood-cells.  This  leads  him  to  the  following  conclusion:  "From  the 
facts  mentioned  it  can  readily  be  seen  that  the  haemolytic  side-chains 
of  the  guinea-pig  are  already  physiologically  present  in  the  spleen  and 
lymph-glands  and  that  the  injection  of  dog  blood  aids  their  hyper- 
production." 

Klein  prepared  the  organ  extracts  by  crushing  them  with  quartz 
gravel,  then  mixing  with  an  equal  amount  of  physiological  salt  solu- 
tion and  filtering  in  the  cold.  The  only  constant  effect  was  the 
haemolytic  action  of  the  extract  of  pancreas;  in  a  few  cases  the  ex- 
tract of  kidney  and  of  intestinal  mucosa  also  dissolved  the  red  blood- 
cells. 

Metchnikoff's  experiments  were  continued  in  his  laboratory  by 
Tarassevitsch,  who  studied  principally  the  organs  of  guinea-pigs, 
rabbits,  and  dogs.  Corresponding  to  Metchnikoff's  first  experiments, 
he  tested  the  haemolytic  action  mostly  on  avian  blood-cells,  but  also 
on  those  of  mammals.  In  the  guinea-pig,  in  the  great  majority  of 
cases,  he  found  the  extracts  of  omentum,  mesenteric  lymph-glands, 
and  spleen  to  be  haemolytic.  Besides  this  pancreas  extract  and  in 
many  cases  salivary  gland  extract  were  haemolytic.  In  general  the 
haemolytic  action  of  the  organ  extracts  of  rabbits  is  weaker  than  that 
from  the  organs  of  guinea-pigs.  Omentum,  spleen,  and  mesenteric 
glands  frequently  were  haemolytic;  the  salivary  glands  acted  feebly; 
bone  marrow,  liver,  and  thymus  were  not  haemolytic.  According  to 
Tarassevitsch,  therefore,  only  the  macrophagic  organs  and  the  digestive 
glands  possess  a  haemolytic  action. 

1  Shibayama,  Centralblatt  f.  Bact.,  Vol.  30,  1901,  No.  21. 
7  Klein,  K.  k.  Ges.  der  Aerzte  in  Wien,  Sitzung  von  Dec.  20,  1901,  reported 
in  Wiener  klin.  Wochenschr.  1901,  No.  52. 

3  Tarassevitsch,  Sur  les  Cytases,  Annal.  de  1'Inst.  Past.  1902. 


THE  H^MOLYTIC  PROPERTIES  OF  ORGAN  EXTRACTS.     269 

If  the  organ  extracts  are  heated  to  56°  C.  for  half  or  one  hour 
the  hsemolytic  property  disappears  in  many  cases;  in  other  cases  it 
is  diminished;  very  rarely  it  remains  unchanged.  According  to 
Tarassevitsch,  this  variation  from  the  "cytases"  (which  in  general 
are  destroyed  by  heating  for  half  an  hour  to  56°  C.)  is  only  an  apparent 
one.  In  the  organ  extracts  the  "  macrocy tase "  is  not  completely 
liberated,  but  is  held  back  to  a  great  extent  by  the  cell  detritus  pres- 
ent in  the  emulsion.  It  leaves  the  detritus  only  very  slowly  and 
incompletely,  as  is  shown  by  the  fact  that  the  entire  emulsion  is  always 
more  active  than  the  fluid  portion  obtained  by  centrifuging,  and 
also  that  by  filtering  through  paper  the  clear  fluid  is  deprived  of  the 
greater  part  of  the  properties  which  the  entire  emulsion  possesses. 
This  filtered  fluid,  in  which,  according  to  Tarassevitsch,  all  the 
"cytases"  present  are  in  dissolved  form,  is  said  to  behave  toward 
thermal  influences  like  haemolytic  serum. 

Finally  according  to  Tarassevitsch  the  thermostability  of  the  entire 
extracts  is  not  very  great.  If  he  heated  his  extracts  a  little  higher,  one 
to  two  hours,  to  58.5°,  60°,  62°,  the  hcemolytic  property  disappeared  com- 
pletely. 

From  this  behavior  toward  thermic  influences  Tarassevitsch 
concludes  that  the  relationship  of  the  haemolytic  substances  of  the 
organ  extracts  to  the  "cytases"  of  serum  is  perfectly  clear,  and  that 
it  is  incorrect  to  ascribe  a  haemolytic  property  which  can  be  de- 
stroyed at  such  low  temperatures,  to  osmotic  phenomena  or  to  the 
presence  of  "de  quelques  substances  chimiques."  Hence,  as  Metchni- 
koff  assumed,  the  organs  in  question  contain  a  macrocy  tase,  and  this 
circumstance  proves  that  the  macrophagic  organs  must  play  a  role  in 
the  formation  of  the  natural  and  the  artificial  haBmolysins. 

In  the  following  pages  we  shall  describe  certain  experiments  in 
which  we  have  reached  essentially  different  results  from  those  obtained 
by  Metchnikoff  and  Tarassevitsch. 

The  emulsion  of  the  organs  was  prepared  as  follows:  The  organs  removed 
from  the  exsanguinated  animals  are  rubbed  up  very  finely  with  sea-sand  which 
has  first  been  purified  with  hydrochloric  acid.  Then  5  to  10  times  their  weight 
of  physiological  salt  solution  is  added  and  the  mixture  thoroughly  shaken 
in  a  shaking-machine  for  two  hours,  whereupon  the  coarser  particles  are  re- 
moved through  several  hours'  centrifuging.  A  more  or  less  uniformly  clouded 
fluid  remains.  The  organ  extracts  were  employed  as  fresh  as  possible,  though 
it  was  found  that  they  could  well  be  preserved  by  freezing  them  at  —10°  to 
-15°C.1 

1  On  thawing  them  out   we  often  observed  the  appearance  of  numerous 


270  COLLECTED  STUDIES  IN  IMMUNITY. 

In  studying  the  haemolytic  action  blood-cells  were  used  which  had  been 
freed  from  serum  as  much  as  possible. 

The  series  of  tubes  was  kept  in  the  thermostat  at  37°  C.  for  two  to  three  hours 
and  overnight  in  the  refrigerator  at  8°  C.  In  the  presence  of  large  amounts  of 
organ  extracts  haemolysis  proceeds  rapidly;  with  small  amounts  it  is  very  slow. 
The  tubes  must  be  frequently  shaken  while  being  kept  at  37°;  the  result  can 
only  be  judged  of  on  the  following  day. 

To  begin  we  sought  to  gain  a  general  idea  of  the  hsemolytic  action 
of  several  organ  extracts  on  various  species  of  blood.  The  extracts 
of  intestine  and  of  stomach  of  the  mouse  as  well  as  that  of  the  stomach 
of  guinea-pigs  and  of  the  pancreas  of  oxen  always  showed  a  strong 
hsemolytic  action  on  all  species  of  blood  which  we  examined,  1.0  cc. 
to  0.5  cc.  of  the  extracts  sufficing  to  completely  dissolve  1  cc.  5% 
blood  of  rabbit,  guinea-pig,  mouse,  rat,  goat,  sheep,  ox,  pig,  horse, 
dog,  or  goose.  The  rest  of  the  organ  extracts  examined,  namely 
guinea-pig  intestine,  rat  intestine,  rat  stomach,  varied  in  their  hremo- 
lytic  property  with  different  bloods,  qualitatively  as  well  as  quanti- 
tatively. Extract  of  guinea-pig  spleen  dissolved  only  dog  blood  and 
guinea-pig  blood ;  extract  of  mouse  spleen  possessed  a  feeble  haBmoly  tic 
action  on  guinea-pig  blood  and  pig  blood.  Extract  of  guinea-pig 
adrenals  dissolved  both  the  blood  species  examined  in  this  case,  viz., 
guinea-pig  blood  and  goose  blood.  We  found  the  extract  of  spleen, 
mesenteric  lymph  nodes,  pancreas,  stomach,  intestine,  and  adrenals 
of  one  dog  to  be  strongly  haemolytic  for  guinea-pig  blood,  whereas 
in  another  case  the  spleen  showed  Itself  absolutely  inert,  although 
the  pancreas  was  strongly  haemolytic.  This  variation  in  the  haBmo- 
lytic  action  on  various  blood-cells  has  already  been  noticed  by  other 
investigators,  and  we  therefore  desire  merely  to  call  attention  to  a 
point  which  thus  far  has  not  been  regarded,  namely,  that  the  organ 
extracts  are  able  to  dissolve  the  blood-cells  of  the  same  species  and  even 
of  the  same  individual  from  which  they  are  derived. 

Thus  according  to  our  experience  emulsions  of  guinea-pig  stomach, 
spleen,  adrenal,  kidney,  and  intestine,  of  mouse  intestine  and  stomach, 
of  rat  intestine  and  stomach,  of  ox  pancreas,  dissolve  the  red  blood- 
cells  of  their  own  species.  The  relation  existing  between  this  action 
on  the  blood  of  the  same  species  and  haemolysis  of  foreign  species  of 
blood  is  shown  by  the  following  two  experiments.  (See  Table  I.) 

clumps  in  the  organ  extracts  which  before  had  been  free  from  visible  particles. 
These  clumps  could  be  separated  by  centrifuge,  and  exhibited  a  heemolytic 
action  when  suspended  in  salt  solution. 


THE  H^MOLYTIC  PROPERTIES  OF  O&GAN  EXTRACTS.     271 


TABLE  I. 

EMULSION  OF  MOUSE  INTESTINE  (10%). 


1  cc.  5% 
Ox  Blood. 

1  cc.  5%  Guinea- 
pig  Blood. 

1  cc.  5%  Mouse 
Blood. 

1.0 

complete 

complete 

complete 

0.75 

0.5 

almost  complete 

tt 

0.35 

trace 

0.25 

0 

trace 

0.2 

0 

0 

0.15 

0 

0 

EMULSION  OF  BEEF  PANCREAS  (10%). 

1  cc.  5% 
Rabbit  Blood. 

1  cc.  5%  Guinea- 
pig  Blood. 

1  cc.  5% 
Ox  Blood. 

0.5 

complete 

complete 

complete 

0.35 

0 

0 

0.25 

strong 

0 

0 

0.15 

0(?) 

0 

0 

These  experiments  show  that  the  susceptibility  of  the  body's  own 
blood  may  be  very  great,  even  as  great  as  that  of  a  foreign  species 
of  blood.  Whether  all  these  extracts  dissolve  the  blood  of  the  own 
individual  we  have  not  determined;  we  regard  it  as  probable,  however, 
since  positive  results  were  obtained  in  all  experiments  which  we  made 
in  this  direction,  especially  with  extracts  of  mouse  intestine  and  of 
guinea-pig  stomach. 

These  experiments  (especially  those  with  the  extract  of  guinea- 
pig  spleen,  which  Shibayama  too  found  to  be  active  only  for  dog 
blood)  show  that  we  are  not  here  dealing  with  hcemolytic  poisons  of  a 
general  kind  (such  as  saponin,  the  gallic  acid  salts,  and  certain  alka- 
loids, like  solanin,  which  dissolve  all  blood-cells  regardless  of  species)  r 
but  that  these  hsemolytic  poisons  possess  a  certain  specificity  which 
is  of  special  biologic  interest. 

The  property  of  organ  extracts  to  dissolve  the  blood-cells  from  the 
same  individual  is  of  great  significance  because  neither  when  normal 
nor  after  immunizing  procedures  does  the  blood-serum  of  these  animals 
ever  contain  substances  which  damage  the  blood-cells  of  the  animal 
itself  (autohsemolysins).  Tarassevitsch  himself  noticed  the  great  dif- 
ference existing,  on  the  one  hand,  between  the  absence  of  a  marked 
haemolytic  action  of  guinea-pig  serum  on  foreign  species  of  blood 
and  the  strong  haBmolytic  action  of  the  extracts  of  certain  guinea-pig 


272  COLLECTED  STUDIES  IN  IMMUNITY. 

organs,  on  the  other.  He  believes  to  explain  this  by  assuming  a 
difference  in  the  macrocytase  extracted  from  the  organs  and  that 
present  in  the  serum.  In  any  case  this  constitutes  a  serious  dilemma 
for  Tarassevitsch;  for  either  there  are  several  "macrocytases"  as 
opposed  to  the  Unitarian  view  of  Metchnikoff  or  the  macrocytase  of 
serum  is  identical  with  that  of  the  organ  extracts.  In  view  of  this 
entirely  different  behavior,  however,  the  latter  does  not  appear 
acceptable  to  Tarassevitsch. 

Our  first  question  was  an  entirely  different  one,  for  in  all  the  cases 
of  haemolysis  and  bacteriolysis  sufficiently  examined  we  had  never 
met  with  a  simple  alexin  in  the  sense  of  Buchner  and  Metchnikoff,  but 
invariably  found  a  coaction  of  amboceptor  and  complement.  In  view 
of  this  our  investigations  had,  above  all,  to  determine  whether  the 
hsemolytic  organ  extracts  could  be  shown  to  be  characterized  by 
complement  and  amboceptor. 

These  first  doubts,  namely,  whether  these  substances  corresponded 
to  what  we  conceive  as  the  complex  haemolysins  of  blood-serum,  led 
us  to  study  the  hsemolytic  organs  in  respect  to  those  main  character- 
istics which  we  have  come  to  know  in  our  study  of  the  complex  hae- 
molysins. These  are:  1.  The  behavior  toward  thermic  influences. 
2.  The  behavior  when  bound  to  the  red  blood-cells  at  low  tempera- 
tures. 3.  The  power  of  producing  antibodies  by  immunization. 

We  shall  begin  by  describing  a  number  of  typical  experiments  which 
show  the  behavior  of  the  organ  extracts  toward  higher  temperature. 
Let  us  glance  first  at  the  experiments  dealing  with  the  effect  of  organ 
extracts  on  goose  blood-cells,  for  this  is  the  blood  species  which  has 
been  mainly  used  by  Metchnikoff  and  Tarassevitsch.  (See  Table  II.) 

These  experiments  clearly  show  that  in  most  of  the  cases  the 
haemolytic  action  of  organ  extracts  on  goose  blood-cells  is  not  at 
all  or  but  slightly  affected  by  a  three-hour  heating,  to  62°  C.,  and  that 
heating  to  100°  C.  for  one  hour  and  even  for  three  hours  does  not 
produce  any  further  damage.  Only  the  hsemolytic  effect  of  extract 
of  mouse  intestine  is  reduced  to  about  one-half  by  the  heating  to 
62°  C. ;  heating  to  100°  C.  for  three  hours  causes  but  little  additional 
damage.  But  that  this  cannot  be  a  true  destruction  of  part  of  the 
hsemolysin  will  be  discussed  later.  We  wish  next  to  present  additional 
experiments  dealing  with  the  behavior  of  heated  organ  emulsions  on 
guinea-pig  blood.  (See  Table  III.) 

Nor  is  this  result  changed  if  stronger  agents,  such  as  alkalies  or 
acids,  are  employed  at  high  temperatures.  (See  Table  IV.) 


THE  H^MOLYTIC  PROPERTIES  OF  ORGAN  EXTRACTS.     273 


TABLE  II. 

A.  ACTION  OP  HEATED  ORGAN  EXTRACTS  ON  GOOSE  BLOOD-CELLS  (1  cc.  5%). 
I.  Extract  of  Dog  Spleen  (10%). 


Not  Heated. 

3  Hra.  (62°). 

0.2 
0.15 
0.1 

complete  solution 

<<              « 

very  little 

complete  solution 
almost  complete 
very  little  to  trace 

II.  Extract  of  Dog  Stomach  (10%). 


Not  Heated. 

3  Hrs.  (62°). 

1  Hr.  (100°). 

3  Hra.  (100°). 

0.35 
0.25 
0.15 
0.1 

complete 

« 

<  < 
very  little 

complete 
<  t 
very  little 

complete 
t  f 
very  little 

complete 
ft 

n 

very  little 

III.  Extract  of  Dog  Pancreas  (10%). 


Not  Heated. 

3  Hrs.  (62°). 

1  Hr.  (100°). 

3  Hrs.  (100°). 

0.75 
0.5 

complete 

14 

complete 

«  < 

complete 

« 

complete 
fairly  complete 

0.35 

strong 

0 

0 

0 

0.25 

very  little 

0 

0 

0 

0.15 

0 

0 

0 

0 

IV.  Extract  of  Dog  Mesenteric  Lymph  Glands  (10%). 


Not  Heated. 

3  Hrs.  (62°). 

1  Hr.  (1006).1 

3  Hrs.  (100°).i 

0.75 
0.5' 
0.35 

complete 
a 

(  ( 

complete 
ft 

almost  complete 

complete 
strong 
very  little 

complete 
strong 
very  little 

1  Enormous  coagula. 
V.  Extract  of  Mouse  Intestine  (5%). 


Not  Heated. 

3  Hrs.  (62°). 

1  Hr.  (100°). 

3  Hre.  (100°). 

0.35 
0.25 
0.2 
0.15 
0.1 

complete 

<  t 
it 

almost  complete 

complete 

strong 
moderate 
little 

complete 

strong 
little 
trace 

complete 

moderate 

t  t 

little 
trace 

274  COLLECTED  STUDIES  IN  IMMUNITY. 

TABLE  III. 

ACTION  OF  HEATED  ORGAN  EXTRACTS  ON  GUINEA-PIG  BLOOD  (1  cc.  5%). 
I.  Extract  of  Dog  Mesenteric  Glands  (5%). 


Not  Heated. 

1  Hr.  (64°). 

30  Hrs.  (100°). 

0.25 
0.15 
0.1 
0.075 

complete 

« 

trace 
0 

complete 

0 
0 

complete 
<  < 

faint  trace 
0 

II.  Extract  of  Ox  Pancreas  (10%). 

Not  Heated. 

1  Hr.  (62°). 

0.35 
0.25 
0.15 

complete 
strong 

complete 
strong 

III.  Extract  of  Ox  Pancreas  (20%). 

Not  Heated. 

1  Hr.  (68°). 

H  Hrs.  (100°). 

0.15 
0.1 
0.075 
0.05 

complete 

<  < 

trace 
0 

complete 

« 

trace 
faint  trace 

complete 

«  < 

trace 
0 

IV.  Extract  of  Guinea-pig  Stomach  (10%). 

Not  Heated. 

3  Hrs.  (65°). 

0.25 
0.2 
0.15 

complete 
strong 

complete 
strong 

TABLE  IV. 
EXTRACT  OF  Ox  PANCREAS  (10%). 


Not  Treated. 

Containing  1/50  n.  HC1 
Heated  to  60°  for  30  Min. 
and  Neutralized 

Containing  1/50  n   NaOH 
Heated  to  60°  for  30  Min. 
and  Neutralized. 

0.35 
0.25 
0.15 
0.1 

complete 

a 

faint  trace 
0 

complete 
almost  complete 
0 
0 

complete 
almost  complete 
0 
0 

THE  H^MOLYTIC  PROPERTIES  OF  ORGAN  EXTRACTS.     275 

All  these  experiments  show  that  the  organ  extracts  will  bear 
heating  to  62-68°  C.  for  hours,  and  even  100°  for  several  hours,  with- 
out suffering  any  change  in  their  haemolytic  properties  worth  men- 
tioning. In  these  experiments,  in  fact,  we  have  been  unable  thus 
far  to  find  any  limit  for  the  thermostability  of  the  organ  extracts. 
We  are  therefore  dealing  with  substances  which  withstand  boiling 
(coctostabile) ,  and  this  fact  in  itself  is  sufficient  to  disprove  the  assump- 
tion that  they  are  "cytases." 

The  next  question,  of  course,  is  how  such  a  fundamental  divergence 
between  our  results  and  those  from  Metchnikoff's  highly  esteemed 
laboratory  can  be  explained.  We  think  we  have  discovered  the  cause 
of  this  difference.  It  is  as  follows: 

In  the  above  experiments  it  is  of  the  greatest  importance  to  shake 
the  fluid  previous  to  testing  its  hsemolytic  property;  in  that  way  the 
more  or  less  plentiful  precipitate  formed  on  heating  is  again  uniformly 
distributed  throughout  the  fluid.  Only  the  coagulum  produced  by 
heating  possesses  a  haemolytic  action.  According  to  our  experience, 
if  a  precipitate  has  been  produced  through  heating,  the  clear  -fluid 
which  is  separated  from  this  no  longer  possesses  any  haemolysin  what- 
ever. If  the  precipitate  is  separated  by  centrifuge  the  clear  fluid  will 
be  found  inert;  on  suspending  the  sediment  in  the  requisite  quantity 
of  physiological  salt  solution  a  new  emulsion  is  obtained  which  has 
preserved  the  haemolytic  property.  This  is  shown  in  the  following 
table.1 

According  to  these  experiments  it  would  seem  very  probable  that 
the  contradictory  results  obtained  by  us  on  the  one  hand  and  by 
Metchnikoff  and  Tarassevitsch  on  the  other  are  due  to  insufficient 
regard  being  paid  by  the  latter  to  the  precipitates  formed  in  the 
organ  extracts  on  heating. 

If  we  assume  that  the  haemolytic,  coctostable  substance  is  present 


1  The  coagula  formed  on  heating  may  be  so  plentiful  that  they  render  an 
exact  observation  of  haemolysis  exceedingly  difficult.  It  is  frequently  seen  that 
haemolysis  by  means  of  heated  organ  extracts  which  are  filled  with  coagula 
proceeds  very  slowly;  apparently  the  precipitates  offer  considerable  resistance 
to  the  escape  of  the  haBmolytic  substance.  Naturally,  this  constitutes  a  source 
of  error,  since  with  low  temperature  and  too  short  a  time  for  observation  the 
haemolytic  action  is  underrated.  This  may  also  explain  the  occasional  weaken- 
ing of  heated  organ  extracts,  to  which  we  have  already  referred;  in  that  case 
the  weakening  would  not  be  due  to  a  partial  destruction  of  the  haemolytic 
substance. 


276 


COLLECTED  STUDIES  IN  IMMUNITY. 

TABLE  V. 

I.  EXTRACT  OF  DOG  LYMPH  GLANDS  (10%). 
Guinea-pig  blood  (1  cc.  5%). 


Fresh. 

1  Hr.  (62°). 
(No  Coagulum.) 

1  Hr.  (100°). 
Slight  Precipitate, 
Centrifuged,  and 
Suspended  in  Salt 
Solution. 

1  Hr.  (100°). 
The  Clear  Fluid 
obtained  by 
Centrifuging. 

2.0 
1.5 

— 

— 

complete 

0 
0 

1.0 

complete 

complete 

<  t 

0 

0.75 

1  t 

<  < 

— 

0 

0.5 

it 

<  i 

complete 

0.25 

i  i 

strong 

— 

— 

0.15 

strong 

very  little 

— 

— 

II.  EXTRACT  OF  DOG  PANCREAS  (20%). 
Guinea-pig  blood  (1  cc.  5%). 


Fresh. 

1  Hour  (62°). 
(No  Coagulum.) 

1  Hr.  (100°). 
Slight  Precipitate. 
Centrifuged,  and 
Suspended  in  Salt 
Solution. 

1  Hr.  (100°). 
The  Clear  Fluid 
obtained  by 
Centrifuging. 

2.0 

— 

— 

complete 

0 

1.5 

— 

— 

a 

0 

1.0 

complete 

complete 

n 

0 

0.75 

i  t 

1  1 

— 

0 

0.5 

n 

little 

moderate 

0 

0.25 

little 

0 

— 

— 

0.15 

<  < 

0 

— 

— 

III.  EXTRACT  OF  DOG  INTESTINE  (10%). 
Goose  blood  (1  cc.  5%). 


1  Hr.  (100°). 

1  Hr.  (100°). 

1  Hr.  (100°). 

Precipitate  again 
Uniformly 
Distributed. 

Precipitate  after  Centri- 
fuging, Suspended  in 
Salt  Solution. 

Centrifuged  Fluid 
still  somewhat 
Cloudy. 

1.5 

complete 

complete 

little 

1.0 

(  t 

«  t 

trace 

0.75 

1  1 

«  « 

0 

0.5 
0.35 

1  1 
almost  complete 

almost  complete 
0 

0 
0 

0.25 

— 

— 

0 

0.2 

— 

— 

0 

0.15 

— 

— 

0 

THE  H^MOLYTIC  PROPERTIES  OF  ORGAN  EXTRACTS.     277 


TABLE  V— Continued. 

IV.  EXTRACT  OF  MOUSE  INTESTINE  (10%). 

Goose  blood  (1  cc.  5%). 


3  Hrs.  (100°). 
Precipitate  again 
Uniformly  Distributed. 

3  Hrs.  (100°). 
Precipitate  Suspended 
in  Salt  Solution. 

3  Hrs.  (100°). 
Clear  Centrifuged  Fluid. 

1.0 

complete 

— 

0 

0.75 

complete 

— 

0.5 

1  1 

<< 

0 

0.35 

1  1 

strong 

0 

0.25 

moderate 

trace 

0.2 

i  ( 

<  i 

— 

0.15 

little 

faint  trace 

— 

0.1 

very  little 

minimal 

— 

in  the  organ  extracts  in  dissolved  form  we  find  it  difficult  to  under- 
stand the  fact  that  it  is  abstracted  from  the  fluid  by  means  of  the 
coagulum  formed  on  heating.  To  be  sure,  one  could  think  of  an 
absorption  by  the  coagulum.  The  complete  abstraction  by  means 
of  heating  is,  however,  readily  understood  if  the  hsemolytic  substance 
is  present,  not  in  solution,  but  in  a  state  of  finest  suspension;  for  it  is 
a  matter  of  common  experience  that  substances  finely  suspended  in 
a  fluid  are  carried  down  with  a  precipitate  produced  in  the  fluid. 
The  technique  of  clearing  cloudy  fluids  rests  to  a  large  extent  on  such 
precipitations. 

We  have  not  yet  been  able  to  decide  definitely  whether  the  ha> 
molytic  substance  is  present  in  the  fluid  in  dissolved  form  or  in  very 
fine  suspension;  we  incline  strongly  to  the  latter  view.  We  base 
this  (1)  on  numerous  experiences  which  show  that  by  filtering  the 
organ  extracts  through  porous  filtering  candles  the  fluid  obtained 
is  entirely  inert;  (2)  on  the  behavior  of  the  h«molytic  substance 
when  treated  with  alcohol. 

One  part  of  a  1%  extract  of  ox  pancreas  is  mixed  with  ten  parts 
96%  alcohol,  and  after  a  time  the  fluid  is  filtered  off  from  the  flaky 
precipitate  which  has  formed.  The  entirely  clear  filtrate  is  distilled 
in  vacuo  and  the  portion  left  behind  mixed  with  physiological  salt 
solution.  A  coarsely  flocculent  suspension  is  thus  obtained  which 
possesses  strong  haemolytic  action,  about  one-half  to  one- third  of  the 
original  strength.  If  this  mixture  is  now  filtered,  the  clear  .filtrate  is 
found  to  be  absolutely  inert,  whereas  the  flakes  washed  from  the 
filter  exhibit  almost  the  full  hsemolytic  effect.  The  following  experi- 
ment will  serve  as  an  example. 


278 


COLLECTED  STUDIES   IN   IMMUNITY 
TABLE  VI. 


GUINEA-PIG  BLOOD  (1  cc.  5%),  EXTRACT  OF  Ox  PANCREAS  (10%).  PORTION 
LEFT  FROM  THE  ALCOHOLIC  DISTILLATE  SUSPENDED  IN  0.85%  SALT  SOLU- 
TION. 


Total  Fluid. 

Clear  Filtrate 

Suspension  of  the 
Flakes. 

1.0 

complete 

0 

complete 

0.5 

'  ' 

0 

'  ' 

0.35 

— 

0 

" 

0.25 

complete 

0 

i  i 

0.15 

— 

0 

strong 

0.1 

moderate 

0 

trace 

We  are  therefore  evidently  dealing  with  a  substance  which  in  the 
above  treatment  is  dissolved  in  the  alcoholic  fluid  but  which  is  soluble 
to  only  a  very  slight  degree  in  salt  solution. 

Naturally  a  certain  degree  of  solubility  is  always  one  of  the  con- 
ditions of  the  haBmolytic  action  observed,  but  this  need  only  be  a 
minimal  one.  The  blood-cells  can  anchor  the  amount  of  hsemolytic 
substance  in  solution  at  any  given  time  and  so  render  the  fluid  capable 
of  taking  up  small  amounts  of  the  substance  anew.  This  conception 
of  a  relative  insolubility  of  the  substance  is  readily  reconciled  with 
the  hsemolytic  action.  The  process  which  takes  place  reminds  one 
of  that  occurring  with  certain  dyes,  which,  although  not  given  off  to 
the  water  from  the  dyed  fibre,  are  nevertheless  able  by  means  of  the 
watery  medium  to  go  from  the  dyed  to  undyed  fibres. 

The  coctostability  of  the  haBmolytic  substances  of  organ  extracts, 
their  adherence  to  solid  particles,  their  solubility  in  alcohol — all  these, 
in  our  opinion,  show  that  these  substances  cannot  be  classed  as  iden- 
tical either  with  the  "cytases"  of  Metchnikoff  or  with  our  complex 
hsemolysins.  Nevertheless  we  have  still  further  examined  these 
substances  for  properties  which  characterize  the  hsemolysins. 

In  one  case,  therefore,  we  studied  the  action  of  our  organ  emulsion 
on  blood-cells  at  0°  C.  in  order  to  determine  the  possiblity  of  separating 
a  possible  amboceptor  and  complement. 

To  each  1  cc.  of  a  5%  suspension  of  guinea-pig  blood  which  had 
been  thoroughly  cooled  on  ice,  varying  amounts  of  cooled  extract  of 
ox  pancreas  were  added  and  the  mixture  kept  at  0°  for  two  hours 
and  frequently  shaken.  In  this  case  slight  solution  occurred  only 
with  large  quantities  of  the  extract.  Then  the  mixtures  were  cen- 
trifuged,  the  sediment  resuspended  in  salt  solution  (1.5  cc.),and  the 


THE  H^MOLYTIC  PROPERTIES  OF  ORGAN   EXTRACTS.     279 

decanted  fluid  mixed  with  0.05  cc.  of  guinea-pig  blood  freed  from 
serum.     (See  Table  VII.) 

TABLE  VII. 

GUINEA-PIG  BLOOD  (1  cc.  5%). 


Pancreas 
Extract. 

Solution  at  the 
End  of  Two 
Hours  at  0°. 

Haemolysis  with 
the  Decanted 
Fluid. 

Haemolysis  of 
Sediment, 

Control,   Absolute 
Action  in 
Warmth. 

1.0 

little 

complete 

complete 

complete 

0.5 

0 

0 

*  ' 

0.35 

0 

0 

<  < 

«  « 

0.25 

0 

0 

almost  complete 

1  ( 

0.15 

0 

0 

strong 

strong 

We  see,  therefore,  that  at  0°  the  single  solvent  dose  has  been  com- 
pletely anchored  by  the  blood-cells  and  that  after  centrifuging  this 
leads  to  complete  solution  at  higher  temperatures;  double  the  solvent 
dose  is  still  completely  anchored  by  the  blood-cells.  This  condition 
of  affairs  does  not  at  all  correspond  to  the  behavior  of  the  complex 
haemolysis  of  serum. 

It  still  remained  to  study  another  fundamental  characteristic, 
namely,  the  formation  of  antibodies. 

We  made  peritoneal  injections  into  rabbits,  using  for  this  purpose 
a  strongly  active  extract  of  ox  pancreas  that  had  been  sterilized  by 
heating  to  60°  C.  for  one  hour.  The  precipitate  which  developed  being 
regarded  as  the  true  active  constituent,  the  mixtures  were  thoroughly 
shaken  and  the  whole  injected.  Two  rabbits  received  20  cc.,  45  cc., 
and  60  cc.  of  the  extract  at  suitable  intervals  and  were  bled  ten  days 
after  the  last  injection.  The  antihaBmolytic  action  of  the  serum  against 
the  extract  was  found  to  be  exactly  the  same  as  that  of  normal  rab- 
bits. (See  Table  VIII.) 

As  can  be  seen  from  this  experiment  (the  result  of  which  is  con- 
firmed by  a  number  of  similar  experiments  with  the  serum  of  other 
rabbits  and  of  a  goat  treated  in  like  manner)  it  has  not  been  possible 
to  produce  antibodies  by  injections  of  pancreas  extract. 

The  experiment,  moreover,  shows  that  normal  rabbit  serum  already 
possesses  a  marked  inhibiting  action  on  the  hamolysis  through  organ 
extracts.1  We  have  been  able  to  demonstrate  this  on  all  the  species 

1  This  action  of  the  serum  must  always  be  taken  into  account  in  the  ex- 
periments, and  the  blood-cells  first  washed. 


280 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE  VIII. 

1  cc.  GUINEA-PIG  BLOOD +0.5  EXTRACT  OF  Ox  PANCREAS  =  TWICE 
THE  SOLVENT  DOSE. 


1.  Of  Rabbits 

+  Serum. 

Immunized  with 

2.  Of  Normal 

Pancreas  Extract 

Rabbits  Inactive. 

cc. 

Inactive. 

1 

0.25 

0 

0 

2 

0.2 

0 

0 

3 

0.15 

0 

0 

4 

0.1 

complete 

almost  complete 

5 

0.075 

1  t 

complete 

6 

0.05 

<  < 

«  « 

7 

0.015 

1  1 

of  sera  investigated  by  us;  it  is  especially  marked  in  ox  serum,  as  can 
be  seen  by  the  following  examples.     (See  Table  IX.) 


TABLE  IX. 
1  cc.  5%  Guinea-pig  Blood +0.5  cc.  Extract  of  Ox  Pancreas. 


cc. 

+  Inactive 
Rabbit  Serum. 

+  Inactive  Goat 
Serum. 

1.0 

0 

0 

0.5 

0 

0 

0.25 

0 

0 

0.1 

almost  complete 

0 

0.05 

complete 

strong 

0.025 

•* 

complete 

0 

Guinea-pig  Blood,  1  cc.  5%+  Extract  of  Ox 

Pancreas,  1  cc.  (  =  4  times  the  solvent  dose) 

+  Inactive  Ox  Serum  (J  hour  at  56°  C.). 

0.05 

0 

0.025 

0 

0.01 

strong 

0 

complete 

That  these  antihsemolytic  actions  of  normal  sera  are  not  due  to 
antibodies  in  the  proper  sense  is  shown  by  the  fact  that  this  protective 
action  withstands  the  action  of  high  temperature,  even  100°  C.  This 
is  shown  by  the  following  table. 


THE  H^MOLYTIC  PROPERTIES  OF  ORGAN  EXTRACTS.     281 
TABLE  X, 


1  cc.  5%  Guinea-pig  Blood  +  0.2  cc.  (1  cc.  =  i)  Goat  Serum. 

Extract  of 
Pancreas. 

Goat  Serum  was  Heated  for  1  Hour 

cc. 

at  70°. 

at  100°. 

1 

2 

1.0 
0.75 

complete 
trace 

complete 
faint  trace 

complete 
n 

3 

0.5 

0 

0 

it 

4 

0.35 

0 

— 

(  ( 

5 

0.25 

0 

— 

«  i 

6 

0.15 

0 

— 

faint  trace 

7 

0.1 

0 

— 

0 

8 

0 

0 

~~~ 

0 

The  serum  was  diluted  five  times  with  tap-water  and  after  heating  the 
corresponding  amount  of  salt  was  added. 

This  experiment  shows  that  the  goat  serum,  which  in  amounts  of 
0.2  cc.  almost  completely  neutralizes  three  times  the  solvent  dose  of 
the  emulsion,  does  not  suffer  the  slightest  loss  of  action  even  when 
heated  to  100°  C.  for  one  hour;  that  an  antibody  in  the  proper  sense 
is,  therefore,  not  present. 

Whether  the  coctostable  substance  which  acts  here  is  a  simple 
unit  which  acts  specifically  on  the  haemolytic  substance  of  the  organ 
extract,  or  whether  we  are  dealing  with  a  complex  of  bodies  having 
an  "antireactive"  action,  can  only  be  determined  by  further  investi- 
gations.1 

The  haemolytic  substances  found  in  organ  extracts  and  examined 
by  us  are,  therefore, 

1.  Coctostable; 

2.  Soluble  in  alcohol; 

3.  Not  complex; 

4.  Not  able  to  excite  the  production  of  antibodies. 

This  shows  that  we  are  dealing  with  substances  which  are  entirely 
distinct  from  the  haBmolysins  of  serum  and  which  belong  into  a 
peculiar  class  of  substances  acting  haemolytically. 

1  Analogous  actions  of  coctostable  substances  have  recently  been  observed 
by  Korschun,  who  has  described  a  "pseudo-antirennin"  of  normal  sera 
(Zeitschr.  f.  physiol.  Chemie,  Vol.  36,  Nos..  2  and  3,  1902).  A  thermostable 
substance  inhibiting  the  action  of  urease  has  also  been  recently  described  by- 
Moll  (Hofmeister's  Beitrage,  Vol.  II,  Xos.  7-9). 


"282  COLLECTED  STUDIES  IN   IMMUNITY. 

These  substances  show  a  certain  analogy  to  the  bactericidal 
bodies  obtained  by  Conradi l  in  the  autolysis  of  organs,  since  the 
latter  are  also  coctostable  and  soluble  in  alcohol.  In  contrast  to 
the  former,  however,  Conradi 's  substances  pass  through  porous  niters. 

At  present  it  is  impossible  to  say  whether  these  substances  are 
already  preformed  in  the  living  cell  or  whether  they  originate  only  on 
the  disintegration  of  the  living  protoplasm  either  through  destruction 
of  the  cells  or  through  the  influence  of  the  extracting  agents.  The 
presence  of  amboceptors  and  complements  in  the  living  cell  is  in  no 
way  prejudiced  by  this  demonstration.  In  the  future,  however,  the 
sources  of  error  pointed  out  by  us  must  be  taken  into  account  in 
drawing  conclusions. 

One  thing  must  be  regarded  as  certain,  that  these  experiments 
disprove  the  identity  of  the  hsemolytic  substances  in  question  and 
the  "cytases"  in  the  complements  of  serum. 

1  Conradi,  Beitrage  zur  chem.  Physiol,  in  Pathol.,  Vol.  1,  Nos.  5  and  6, 1901. 


XXVI.   REVIEW  OF  BESREDKA'S  STUDY,  "LES  ANTI- 
HEMOLYSINES  NATURELLES." l 

By  H.  T.  MARSHALL,  M.D.,  and  Dr.  J.  MORGENROTH.* 

THE  chief  result  of  Besredka's  study  is  the  following  conclusion: 
The  serum  of  sick  and  healthy  persons  contains  an  antihcemolysin,  in 
the  form  of  a  simple  antiamboceptor,  which  acts  exclusively  on  the 
specific  ambocepter  fitting  human  blood.  The  amboceptor  used  in 
this  author's  experiments,  and  conceived  as  strictly  Unitarian,  was 
derived  from  a  goat  treated  with  human  blood.  Antihsemolysins 
which  protect  the  blood-cells  of  species  othe*r  than  man  against  hsemoly- 
sins  are  not  present  in  human  serum,  and  the  rule  that  the  normal 
antihsemolysin,  Le.,  the  antiamboceptor  of  a  serum,  always  protects 
only  its  own  blood-cells,  is  of  general  application. 

It  was  easy  for  us  to  show  by  experiments  that  the  last  generaliza- 
tion is  entirely  untenable.  The  most  varied  kinds  of  sera  (thus 
especially  horse  serum)  protect  human  blood-cells  against  specific 
hsemolysins, 3  and  conversely,  according  to  our  experiments,  human 
serum  protects  ox  blood-cells. 

It  is  absolutely  necessary,  above  all,  to  get  the  two  false  premises 
out  of  the  way  which  give  rise  to  all  of  Besredka's  mistakes.  This 
is  a  simple  matter,  for  these  premises  were  possible  only  because  the 
experiments  which  had  long  since  show7n  them  to  be  untenable  were 
ignored.  The  two  erroneous  premises  are: 

1 .  All  the  amboceptors  obtained  by  injecting  any  species  whatsoever 
with  a  particular  species  of  blood  are  entirely  identical.  Thus  Bes- 
redka  assumes  that  if  different  species,  e.g.,  rabbits,  guinea-pigs, 

'•  Annal.  de  1'Institut  Pasteur.  Oct.  1901, 

2  From  a  detailed  study,  '*  Uber  Anticomplemente  und  Antiamboceptoren 
normaler    Sera    und    pathologischer   Exsudate,"    appearing   in   Zeitschrift   fur 
klinische  Medicin,  where  the  experimental  part  is  to  be  found. 

3  See  our  experiments  in  Zeitschr.  fiir  klin.  Medicin,  Table  III. 

283 


284  COLLECTED  STUDIES  IN   IMMUNITY. 

goats,  are  injected  with  blood-cells  of  a  different  species,  say 
the  amboceptors  developed  will  all  be  identical. 

2.  Haemolysis  of  foreign  species  of  blood  by  normal  sera  is  due 
exclusively  to  the  presence  of  a  single,  simple  alexin,  and  not  to  a 
complex  hsemolysin  consisting  of  amboceptor  and  complement. 

The  thorough  studies  of  Ehrlich  and  Morgenroth  positively  prove 
the  incorrectness  of  the  first  assumption.  Above  all,  these  investi- 
gations showed  that  that  body  in  a  haemolytic  immune  serum,  which 
we  term  the  amboceptor,  can,  in  one  and  the  same  animal,  be  shown 
experimentally  to  be  made  up  of  a  host  of  different  kinds  of  amboceptors^ 
Furthermore,  by  means  of  combining  experiments,  of  experiments 
with  an  artificially  produced  antiamboceptor,  and  by  studies  on  the 
complementibility  of  amboceptors  of  different  animal  species  it  was 
shown  that  amboceptors  directed  against  the  same  species  of  blood, 
which  are  obtained  from  different  animal  species,  differ  not  only  in 
their  complementophile  group  but  also  in  their  cytophile  group. 

Besredka,  who  only  learned  of  this  study  after  the  completion  of 
his  own,  regrets  that  "etant  donne  la  complexite  de  plus  en  plus 
grande  de  la  question,  de  ne  pas  pouvoir  suivre  ici  les  auteurs  dans 
leur  argumentations."  It  would  be  deplorable  if  the  principle  should 
gain  ground  that  the  results  of  other  workers  can  simply  be  ignored 
on  the  plea  that  the  verification  of  the  experimental  evidence  is- 
rather  difficult  owing  to  its  complexity.  Finally,  the  diversity  of  the 
amboceptors  has  alread}^  been  established  by  the  studies  on  isolysins.1 
In  this  it  was  shown  that  even  with  twelve  goats  treated  with  goat 
blood,  twelve  different  isolysins  are  to  be  distinguished,  i.e.,  twelve 
amboceptor  complexes  against  the  same  species  of  blood. 

This  large  number  of  amboceptors  fitting  one  blood-cell  corre- 
sponds to  a  like  condition  of  the  blood-cell's  receptors.  These  must 
be  extraordinarily  manifold,  because,  besides  the  receptors  which 
anchor  the  amboceptors,  there  are  present  the  most  varied  receptors 
for  the  numerous  simple  haemolysins  and  haemagglutinins.  This  view, 
enunciated  in  detail  by  Ehrlich,2  has  recently  been  confirmed  by 
the  experiments  of  Landsteiner  and  Sturli.3  These  authors  showed 
that  blood-cells  which  have  been  completely  saturated  with  the 

1  Ehrlich  and  Morgenroth.     (See  page  88.) 

'Ehrlich,  Nothnagels  Spec.  Pathol.  u.  Therapie,  Vol.  VIII,  1901. 
3  Landsteiner  and  Sturli,  Uber  die  Haemagglutinine  normaler  Sera.     Wiener 
klin.  Wochensch.  1902,  No.  2. 


REVIEW  OF  BESREDKA'S  STUDY.  285 

agglutinin  of  one  normal  serum  can  still  take  up  in  succession  the 
agglutinin  of  a  second,  third,  and  even  fifth  serum  in  any  order  one 
chooses.  Thus  the  agglutinin  of  horse  serum  was  still  bound  by 
pigeon  blood-cells  which  had  been  treated  with  goat  and  rabbit 
serum  to  such  an  extent  that  the  cells  were  unable  to  abstract  any 
more  agglutinin  from  these  sera.  These  results  are  only  comprehen- 
sible if  one  assumes  a  large  number  of  different  receptors  for  the 
agglutinins  of  different  sera,  and  it  is  therefore  surprising  to  find  that 
just  these  experiments  which  harmonize  so  well  with  Ehrlich's  views 
should  be  given  a  different  and  complicated  interpretation  by  Land- 
steiner  and  Sturli. 

Besredka's  second  premise  likewise  does  not  correspond  to  the 
facts.  It  is  now  three  years  since  Ehrlich  and  Morgenroth  (see  page 
11)  demonstrated  the  complex  nature  of  normal  hsemolysins  in  a 
number  of  cases;  later  they  brought  forward  evidence  in  favor  of  the 
plurality  of  complements.  In  a  final  study  on  this  subject  Sachs  has 
recently  (see  page  181)  shown  that  in  those  cases  in  which  other 
investigators  did  not  succeed  in  demonstrating  the  complexity  of 
normal  hsemolysins  only  technical  difficulties  and  experimental  errors 
were  to  blame. 

After  this  brief  analysis  of  the  principles  involved,  we  can  pro- 
ceed to  study  Besredka's  experiments  and  discuss  his  conclusions 
from  the  same. 

The  case  especially  investigated  by  Besredka  deals  with  the  com- 
bination human  blood +ambocep tor  of  a  goat  immunized  with  human 
blood  and  guinea-pig  serum  as  complement.  If  inactive  human  serum 
is  added  to  this  combination,  solution  will  be  prevented,  as  we  were 
able  to  verify.  From  this  behavior  of  the  human  serum  Besredka 
concluded  that  this  must  contain  an  antiamboceptor,  giving  the  fol- 
lowing as  his  reasons. 

According  to  Besredka  the  serum  of  each  particular  animal  species 
contains  a  single,  simple  "cytase  "  specific  for  this  animal.  This 
author  has  now  sought  to  determine  whether  human  serum  as  such 
contains  an  "anticytase"  against  the  "cytase"  in  question;  in  other 
words,  whether  in  this  case  the  inactive  human  serum  contains  an 
anticytase  against  guinea-pig  serum.  The  solution  of  this  problem 
was  extremely  easy  for  Besredka.  Guinea-pig  serum,  as  we  know, 
dissolves  certain  species  of  blood,  and  does  so  only  by  means  of  its 
"cytase."  This  action  is  not  inhibited  by  human  serum.  Hence 


286  COLLECTED  STUDIES  IN  IMMUNITY. 

human  serum  contains  no  anticytase  whatever,  and  when,  as  in  the 
above  combination,  human  blood  -f  specific  ambocep tor  +  guinea-pig 
serum,  this  serum  exerts  a  protective  action,  it  follows  by  exclusion 
that  this  action  is  due  to  an  antiamboceptor. 

The  fundamental  error  in  this  method  of  proof  lies,  as  already 
mentioned,  in  the  assumption  of  a  simple  cytase,  which  cytase,  more- 
over, is  able  by  itself  to  effect  haemolysis.  As  a  matter  of  fact,  how- 
ever, solution  of  the  blood-cells  by  guinea-pig  serum  is  brought 
about  only  by  this,  that  the  blood-cells  combine  with  a  normal  am- 
boceptor  present  in  the  blood  serum,  and  that  this  thereupon  anchors 
the  complement  (cytase)  which  effects  solution.  If  the  complement 
in  itself  is  conceived  as  a  single  substance,  one  could  conclude  from 
the  fact  that  the  human  serum  does  not  prevent  this  normal  haemoly- 
sis that  the  human  serum  contains  neither  an  antibody  against  the 
normal  ambocep  tor  nor  against  "the  complement."  In  reality, 
however,  "the  complement"  is  made  up  of  numerous  partial  com- 
plements, one  or  another  of  which  is  dominant  for  the  completion 
of  particular  ambocep  tors,  be  these  haemolytic  or  bacteriolytic.  This 
theory  of  dominant  complements  has  been  firmly  established  by 
Ehrlich  and  Marshall.1 

It  has  already  been  proven  for  anticomplementary  sera  that  such 
a  serum  neutralizes  only  part  of  the  complements  of  a  second  serum, 
not  all.  Marshall  and  Morgenroth 2  have  shown  that  the  anticom- 
plement  of  a  human  ascitic  fluid  prevents  the  complementing  action 
of  guinea-pig  serum  for  one  haemolytic  amboceptor  leaving  that  of 
another  intact. 

Now  Besredka  showed  that  human  serum  does  not  prevent  the 
normal  haemolytic  action  of  a  certain  serum,  although  it  acts  anti- 
haemolytically  when  this  is  used  as  complement  for  an  amboceptor 
produced  by  immunization.  The  only  conclusion  to  be  drawn  from 
this  is  that  the  human  serum  contains  no  anticomplement  which 
acts  against  the  complement  dominant  in  the  case  of  the  normal 
haemolysis.  This,  of  course,  does  not  prevent  the  same  serum  from 
acting  on  other  partial  complements  which  are  dominant  in  other 
cases.  We  see,  therefore,  that  Besredka  Js  entire  method  of  proof 
lacks  a  firm  basis. 

It  is  further  to  be  remembered  that  such  questions  are  to  be  de- 

1  See  page  226  et  seq.  2  See  page  222. 


REVIEW  OF  BESREDKA'S  STUDY.  287 

cided  not  by  pure  speculation  but  by  experimental  means.  The 
centrifugal  method  allows  us  to  demonstrate  antiamboceptor  and 
anticomplement  directly,  as  such,  entirely  independent  of  all  theo- 
retical speculations.  In  the  case  here  described,  we  have  shown 
that  an  anticomplement  action  is  present  almost  exclusively,  com- 
pared with  which  the  slight  antiamboceptor  action  is  of  no  account.1 

As  a  result  of  our  own  results  we  must  maintain,  first,  that  the 
major  part  of  the  anti  action  of  the  human  serum  described  by  Bes- 
redka  is  due  to  the  anticomplement;  second,  that  Besredka's  ex- 
perimental method  allows  no  conclusions  whatever  regarding  an 
anti-immune  body;  and  third,  that  the  part  played  by  the  individual 
factors  in  this  antihsemolytic  action  can  only  be  decided  by  the 
method  employed  by  us. 

After  having,  then,  as  a  result  of  the  experiments  with  human 
blood,  erroneously  ascribed  the  antihsemolytic  action  to  an  antiam- 
boceptor, Besredka  continues  his  study  by  investigating  whether  this 
supposed  antiamboceptor  is  specific,  i.e.,  only  for  human  blood  and 
serum  dissolving  human  blood.  In  this  sense  he  arrives  at  a  posi- 
tive conclusion.  His  generalization  is  based  on  the  following  obser- 
vation: He  finds  that  human  serum  does  not  protect  sheep  blood 
against  the  hsemolytic  serum  of  a  goat  immunized  with  sheep  blood, 
the  haBmolytic  serum  being  reactivated  with  guinea-pig  serum.  We 
have  made  the  same  observation  and  studied  just  this  behavior  by 
means  of  a  human  ascitic  fluid.  The  case  in  question,  however, 
constitutes  a  special  exception,  due  to  a  partial  anticomplement, 
and  it  is,  therefore,  peculiarly  unsuited  as  the  basis  for  a  generaliza- 
tion. Our  experiments  show  that  on  investigating  other  cases, 
human  serum  is  found  to  exert  a  considerable  protection  against 
normal  hsemolysins  and  those  produced  by  immunization  which  dis- 
solve other  species  of  blood — ox  blood  in  our  case.  Here  also,  how- 
ever, this  protection  is  due  to  anticomplements  and  not  to  anti- 
amboceptors,  at  least  so  far  as  can  be  determined  by  an  exact  analysis. 

1  The  destruction  and  weakening  of  the  antihsemolysin  which  Besredka 
shows  occurs  with  longer  heating  to  65-67°  C.  is,  of  course,  in  no  way  char- 
acteristic for  the  nature  and  mode  of  action  of  the  antibody.  We  showed 
that  this  temperature  injures  both  antiamboceptor  and  anticomplement.  Be- 
sides, the  behavior  toward  narrowly  limited  thermal  influences  does  not  possess 
the  significance  of  a  group  reaction.  This  is  well  shown  by  the  occurrence  of 
a  thermostable  complement  (Ehrlich  and  Morgenroth,  page  11)  and  ther- 
molabile  amboceptors  (Sachs,  see  page  181). 


288  COLLECTED  STUDIES  IN  IMMUNITY. 

Finally,  the  fact  that  at  times  a  small  part  of  the  antihsemolytic 
action  (as  in  our  experiments  with  a  human  exudate  and  ox  blood) 
is  due  to  an  antiamboceptor,  removes  the  basis  for  Besredka's  gen- 
eralization that  a  normally  present  antiamboceptor  always  protects 
only  its  own  blood-cells. 

From  all  this  it  follows  that  the  part  believed  to  be  played  by  the 
antiamboceptors  of  human  and  animal  body  fluids  in  the  prevention 
of  hremolysis  is  materially  decreasing  in  favor  of  the  part  taken  by 
the  anticomplement.  There  is  no  doubt  at  all  that  antiamboceptors 
exist  in  normal  serum;  this  was  first  proven  some  time  ago  by  Ehr- 
lich  and  Morgenroth,1  and  also  by  P.  Miiller.2  These  antiambocep- 
tors do  not,  however,  occur  regularly,  as  was  also  pointed  out  at 
that  time. 

Our  analysis  therefore  shows  that  since  the  fundamental  fact 
does  not  apply,  the  extensive  theoretical  conclusions  drawn  by  Bes- 
redka  from  the  exclusive  protection  of  the  homologous  blood-cells  by 
the  serum  cannot  be  recognized.  That  the  amboceptors  present  do 
actually  primarily  protect  the  blood-cells  of  the  corresponding  species 
is  probable  in  itself,  for  according  to  our  view,  as  mentioned  elsewhere,3 
they  represent  free  cell  receptors.  Besredka  assumes  that  the  reason 
for  the  development  of  his  supposed  antiamboceptors  is  this:  that 
blood-cells,  which  are  constantly  dying  in  the  organism,  cause  the 
production  of  hsemolysins.  These  would  endanger  life  if  the  organ- 
ism did  not  paralyze  their  action  through  the  development  of  anti- 
amboceptors. Such  a  regulating  contrivance  can  surely  not  be  very 
common,  since  it  was  not  observed  by  Ehrlich  and  Morgenroth  in  their 
numerous  experiments  on  isolysins,  in  which  it  would  most  readily 
have  been  discovered.  But  if  such  a  contrivance  were  a  necessity, 
it  would  have  to  be  constant.  This,  however,  is  not  at  all  the  case 
as  we  have  already  established.4 

The  simplest  and  most  natural  assumption  is  that  the  antiambo- 
ceptors are  nothing  else  than  products  of  cell  disintegration,  free 
receptors  which  are  capable  of  binding  amboceptors  and  so  exert  a 
deflecting  influence.  The  assumption  that  these  free  receptors  are 
products  of  retrogressive  metabolism  is  borne  out  by  the  fact  estab- 

1  See  page  88  et  seq. 

3  P.  Miiller,  Centralblatt  f.  Bakt.,  Vol.  29,  1901. 

3  Morgenroth.     (See  page  241  et  seq.) 

4  See  pages  23  and  71  et  seq. 


REVIEW  OF  BESREDKA'S  STUDY.  289 

lished  by  Schattenfroh  1  that  they  are  excreted  through  the  urine 
in  considerable  amounts. 

One  reason  above  all  has  led  us  to  believe  that  Besredka's  views 
required  to  be  controverted  in  detail,  namely,  the  fact  that  they 
maintain  the  Unitarian  conception  that  only  one  hsemolysin  is  pos- 
sible against  a  given  species  of  blood  and  one  bacteriolysin  against 
a  given  species  of  bacterium.  This  conception  can  seriously  retard 
the  development  of  the  doctrine  of  immunity  and  especially  of  the 
practical  application  of  this  doctrine. 

The  recent  investigations  which  have  demonstrated  the  exceeding 
multiplicity  of  the  cell's  receptors  and  of  the  amboceptors  obtained 
by  immunizing  with  these  receptors  show  that  this  study  can  be 
successfully  pursued  along  two  directions.  One  of  these  consists  in 
the  production  of  polyvalent  sera  by  immunizing  with  numerous 
strains  of  the  same  bacterial  species.  It  may  be  assumed  that  the 
varieties  of  a  bacterial  species  contain  the  various  receptors  in  very 
varying  proportions,  and  this  is  confirmed  especially  by  Durham's 
experiments  concerning  the  agglutinatibility  of  different  strains  of 
coli  by  specific  sera.  A  sufficient  increase  of  all  the  amboceptor 
types  in  question  is  therefore  possible  only  after  a  high  degree  of 
immunization  has  been  effected  against  a  large  number  of  related 
strains.  This  procedure  had  previously  been  chosen  by  Denys  and 
van  de  Velde  in  the  production  of  their  polyvalent  streptococcus 
serum,  and  has  recently  been  employed  by  Wassermann  and  Oster- 
tag2  for  the  preparation  of  an  effective  serum  against  hog  cholera. 
These  procedures  are  based  entirely  on  the  experiments  of  Ehrlich 
and  Morgenroth,  just  mentioned. 

The  other  method  of  obtaining  effective  bactericidal  sera  is  based 
on  the  assumption  that  the  amboceptors,  according  as  they  are  de- 
rived from  different  animal  species,  differ  from  one  another.  So  far 
as  this  point  is  concerned,  we  may  refer  to  the  statements  of  Ehrlich 
and  Morgenroth,3  which  are  summed  up  in  the  sentence,  "it  would 
therefore  be  advisable  not  to  attempt  the  production  of  bactericidal 
sera  from  a  single  animal  species  as  is  now  customary,  but  to  make 


1  Schattenfroh,  Munch,  med.  Wochensch.  1901,  No.  31. 

2  Wassermann  and  Ostertag,  Monatsch.  f.  prakt.  Thierheilk,  Vol.  13,  foot- 
notes. 

3  See  page  110. 


290  COLLECTED  STUDIES  IN   IMMUNITY. 

a  preparation  containing  a  mixture  of  immune  sera  derived  from 
animals  whose  receptor  apparatus  are  as  divergent  as  possible." 

Practical  results  along  these  lines  have  already  been  achieved 
by  Schreiber,1  who  made  a  hog-cholera  serum  from  horses  and  oxen; 
and  recently  Romer,2  by  paying  attention  to  just  this  point,  has 
obtained  an  effective  pneumococcus  serum  by  utilizing  several  differ- 
ent species  of  animals.  In  view  of  these  attempts  to  apply  the 
above  principles  practically,  it  would  be  regrettable  if  the  untenable 
Unitarian  view  maintained  by  Besredka  were  to  hinder  the  further 
development  of  these  methods. 

1  Schreiber,  Berlin  thierarztl.  Wochenschr.  1902,  No.  19. 

8  Romer,  v.  Graefe's  Archiv  f .  Ophthalmologie,  Vol.  54,  1902. 


XXVII.  THE  MODE   OF  ACTION  OF  COBRA  VENOM.* 

By  PRESTON  KYES,  A.M.,  M.D.,  Associate  in  Anatomy,  University  of  Chicago, 
Fellow  of  the  Rockefeller  Institute  for  Medical  Research. 

I.  Concerning  the  Amboceptors  of  Cobra  Poison. 

THE  most  important  contributions  in  recent  years  to  our  knowl- 
edge of  the  action  of  animal  poisons  are  the  recently  published  in- 
vestigations of  Flexner  and  Noguchi2  on  haemolysis  by  means  of 
snake  venom.  These  authors  have  found  that  although  red  blood- 
cells  whose  serum  has  been  completely  removed  by  washing  with 
salt  solution  are  agglutinated  by  snake  venom,  they  are  not  dissolved. 
If,  however,  serum  is  added  to  the  washed  blood-cells,  or  if  un- 
washed blood  is  usedT  haemolysis  ensues.  From  this  Flexner  and 
Noguchi  conclude  that  the  haBmolytic  action  of  the  snake  venom 
is  due  to  two  factors.  One  of  the  components  is  contained  in  the 
snake  venom  itself,  and  is  said  to  bear  heating  to  about  90°  C.  very 
well.  The  other  component  is  a  constituent  of  the  serum;  to  a 
certain  extent  this  activates  the  poison  which  in  itself  has  no  action. 

Flexner  and  Noguchi  therefore  arrive  at  the  conclusion  that  snake 
venom  is  made  up  of  a  number  of  substances,  acting  after  the  manner 
of  amboceptors,  which  are  activated  by  certain  complements  of  the  serum. 

The  great  significance  of  this  interesting  fact  is  at  once  evident. 
While  formerly  snake  venom  was  regarded  as  a  simple  poison  acting 
after  the  manner  of  toxins,  this  shows  that  the  hsemolytic  action 
of  snake  venom  is  somewhat  more  complex,  being  identical  with 
the  mechanism  of  the  hsemolysins  of  blood  serum,  as  this  has  been 
conceived  by  Ehrlich  and  Morgenroth.  For  this  reason  Flexner 
and  Noguchi's  discovery  was  hailed  with  especial  delight  here  in 
the  Frankfurt  Institute. 


1  Reprint  from  the  Berl.  klin.  Wochenschr.  1902,  Nos.  38  and  39. 

2  S.  Flexner  and  H.  Noguchi,  Snake  Venom  in  relation  to  Haemolysis,  Bacteri- 
olysis, and  Toxicity.     Journ.  of  Exper.  Medicine,  Vol.  VI,  No.  3,  1902. 

291 


292 


COLLECTED  STUDIES  IN  IMMUNITY. 


In  view  of  the  exceeding  importance  of  these  questions  it  seemed 
advisable  to  proceed  from  these  new  facts  and  attempt  to  penetrate 
more  deeply  into  the  mechanism  of  the  snake  venom's  action.  We 
had  at  our  disposal  two  specimens  of  dried  cobra  poison  the  hsemolytic 
strength  of  which  had  proved  to  be  almost  identical  and  for  which 
we  are  indebted  to  Dr.  Lamb  and  Prof.  Calmette. 

A  one  per  cent  solution  of  the  dried  cobra  poison  in  0.85%  salt 
solution  served  as  our  standard  poison.  This  solution  when  kept 
on  ice  was  preserved  unchanged  for  several  days. 

The  experiments  were  made  with  the  following  animal  species: 
man,  ox,  horse,  goat,  sheep,  dog,  rabbit  and  guinea-pig.  Guided 
by  Flexner  and  Noguchi's  observations,  we  at  first  used  only  blood- 
cells  which  had  been  freed  from  serum.  This  was  accomplished 
by  making  a  2J%  suspension  of  the  cells  in  0.85%  salt  solution, 
centrifuging,  decanting  the  fluid,  and  then  adding  anew  the  same 
amount  of  salt  solution.  This  was  always  done  twice  and  then  a 
5%  suspension  was  made. 

All  the  tubes  of  a  given  series  contained  1  cc.  of  a  5%  blood 
suspension  and  they  were  all  made  up  to  the  same  volume  (2  to 
2.5  cc.)  by  the  addition  of  salt  solution.  The  specimens  were  kept 
in  the  incubator  at  37°  C.  for  two  hours,  and  then  placed  on  ice  at 
6°  to  8°  C.  until  the  following  morning. 

According  to  our  experience  there  are  two  kinds  of  blood-cells 
so  far  as  their  behavior  toward  cobra  venom  is  concerned : 

(1)  Those  that  in  themselves  are  dissolved  by  the  cobra  venom. 

(2)  Those  that  are  affected  by  the  cobra  venom  only  after  the 

addition  of  other  substances  (complements,  etc.). 
The  following  table  will  show  the  behavior  of  washed  red  blood- 
cells  of  various  species  toward  cobra  venom: 

TABLE  I. 


Amount 

1  cc.  5%  Blood  Suspension. 

of  Cobra 

Venom, 
cc. 

Guinea- 
pig. 

Dog. 

Man. 

Rabbit. 

Horse. 

Ox. 

Sheep. 

Goat. 

1.0 
0  1 

complete 

complete 

complete 

complete 

complete 

0 

0 

0 

0  05 

' 

almost  complete 

trace 

1 

0.025 

' 

little 

faint  trace 

I 

0  01 

'  ' 

0 

0 

0  005 

' 

No  solution 

0.0025 

almost  complete 

0.001 

little 

strong 

moderate 

0.0005 

trace 

trace 

trace 

0.0001 

0 

0 

0 

THE  MODE  OF  ACTION   OF  COBRA  VENOM.  293 

From  this,  two  groups  of  blood-cells  can  at  once  be  recognized 7 
namely,  blood-cells  like  those  of  guinea-pig,  dog,  rabbit,  man,  and 
horse,  which  are  dissolved  by  the  cobra  venom,  and  blood-cells  which 
are  not  affected  under  these  circumstances  even  with  large  amounts 
of  the  poison.  The  sensitive  blood-cells  do  not  all  possess  the  same 
vulnerability,  but  manifest  considerable  variations,  depending  on 
the  species  to  which  they  belong.  This  is  the  case  with  all  haemolytic 
poisons.  Naturally  besides  this  there  are  certain  individual  fluctua- 
tions in  vulnerablilty.  The  blood-cells  of  the  dog  and  the  guinea- 
pig  are  the  most  sensitive  since  as  a  rule  0.25  cc.  of  a  1: 10,000  dilu- 
tion of  the  poison  still  produces  complete  solution.  The  blood-cells 
of  the  horse  proved  least  sensitive,  for  here  it  required  1.0  cc.  of  a 
1:1000  dilution  of  the  poison  to  produce  solution.  The  difference 
in  vulnerability  is  therefore  one  of  forty  times. 

In  view  of  Flexner  and  Noguchi's  experiments  by  which  the 
amboceptor  character  of  the  haemolytic  portion  of  snake  venoms 
was  demonstrated,  it  seemed  advisable  to  undertake  activating 
experiments  in  those  cases  in  which  the  cobra  venom  did  not  effect 
spontaneous  solution. 

It  was  actually  very  easy  to  produce  solution  by  the  addition  of 
foreign  sera.  We  shall  shortly  show  that  when  the  observations  of 
Calmette  1  are  taken  into  account  these  activities  are  not  all  due  to 
complements.  According  to  our  conception  only  such  substances  are 
complements  which  in  general  are  inactivated  at  a  temperature 
between  52°  and  60°,  in  some  cases  even  somewhat  higher.  This 
corresponds  to  the  greater  or  less  degree  of  lability  of  the  complements 
thus  far  known. 

.  In  our  experiments  such  pure  complementings  were  met  with 
in  the  following  combinations: 

Horse  blood ox  serum 

Ox  blood guinea-pig  serum 

Sheep  blood guinea-pig  serum 

Rabbit  blood guinea-pig  serum 

Table  II  shows  such  an  activation  of  the  cobra  venom.  It  also 
shows  that  the  serum  employed  lost  its  complementing  property 
by  half  an  hour's  heating  to  56°. 

1  A.  Calmette,  Sur  1'action  hemolytique  du  venin  de  cobra.     Comptes  rend, 
de  I'Acade'mie  des  Sciences,  T.  134,  No.  24,  1902. 


294 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE   II. 


Amount  of  the 
Guinea-pig  Serum 
(i)  Added. 

cc. 

1  cc.  5%  Sheep  Blood  + 

a 

Guinea-pig  Serum 
only. 

b 

0.02  cc.  1%  Cobra  Poison  +  Guinea-pig  Serum. 

I. 
Normal. 

II. 
Heated  Half  an  Hour 
to  56°  C. 

0.5 
0.25 
0.1 
0.05 
0.025 
0.01 

little 
trace 
0 
0 
0 
0 

complete 
strong 
little 
trace 
faint  trace 
0 

0 

0 

0 
0 
0 
0 

From  these  experiments  it  can  be  seen  that  in  the  cases  described 
the  cobra  venom  has  the  character  of  an  amboceptor  and  that  the 
amboceptors  are  activated  by  serum  complements  which  possess 
the  ordinary  degree  of  thermolability. 

We  have  thought  it  necessary  to  determine  the  mode  of  action 
of  both  substances  according  to  the  method  used  in  previous  studies 
on  haemolysis.  Hence  we  next  studied  the  behavior  of  sheep  blood- 
cells  toward  the  isolated  cobra  venom  and  toward  the  complement. 
So  far  as  the  behavior  toward  the  poison  alone  is  concerned  it  can  be 
shown  that  this  poison  is  bound  by  the  sheep  blood-cells  although 
these  are  not  by  themselves  dissolved  by  cobra  venom.  This  confirms 
the  statement  of  Flexner  and  Noguchi.  According  to  our  experience, 
however,  the  blood-cells  possess  relatively  feeble  binding  powers, 
especially  in  dilute  solutions  of  the  poison.1  On  the  other  hand 
the  complement  alone  is  not  at  all  bound  by  the  blood-cells.  This 
is  borne  out  by  the  fact  that  at  0°  C.  sheep  blood-cells  are  not  dissolved 
by  cobra  venom + guinea-pig  serum;  while  at  8°  C.  only  a  trace  of 
solution  occurs.  If  a  separation  experiment  is  made,  so  that  ambo- 
ceptor and  complement  are  allowed  to  act  on  blood -cells  at  0°  C., 

1  The  statements  of  Decroly  and  Rousse  (Archiv.  internat.  de  pharma- 
codynamie  et  de  therapie,  Vol.  VI,  1899)  are  in  entire  accord  with  the  slight 
binding  powers  of  red  blood-cells  for  snake  poison.  They  find  that  in  the  animal 
body  also  snake  poison  is  bound  very  much  more  slowly  than  diphtheria  or 
tetanus  poison.  Rabbits  which  had  been  intravenously  injected  with  a  fatal  dose 
of  snake  venom  could  be  saved  even  after  ten  minutes  by  bleeding  and  trans- 
fusing fresh  blood,  whereas  with  diphtheria  or  tetanus  poison,  even  though  the 
same  treatment  was  done  immediately,  the  fatal  ending  could  not  be  averted. 


THE  MODE  OF  ACTION  OF  COBRA  VENOM.  295 

after  which  the  blood  cells  and  fluid  are  separated  by  centrifuge,  it 
will  be  found  that  the  blood-cells  have  taken  up  a  certain  portion 
of  the  amboceptors,  but  none  of  the  complement.  These  experiments 
would  seem  to  prove  the  amboceptor  character  of  the  cobra  poison, 
at  least  for  the  above  cases,  entirely  according  to  the  ideas  of  Flexner 
and  Noguchi. 

II.  Concerning  Endocomplements.1 

We  shall  now  analyze  the  phenomena  which  we  observe  with 
those  blood-cells  which,  like  guinea-pig  blood-cells,  are  directly  dis- 
solved by  cobra  poison.  This  solution  could  be  explained  by  assuming 
that  cobra  poison,  besides  the  amboceptors,  contains  true  toxins 
which  are  analogous  to  the  diphtheria  toxin  and  exert  a  toxic  action, 
i.e.,  effect  haemolysis,  without  the  intervention  of  a  complement. 
In  that  case,  however,  one  would  be  compelled  to  assume  further 
that  only  part  of  the  species  of  blood-cells  react  to  this  poison.  The 
incorrectness  of  this  conception  is  readily  demonstrated. 

The  observation  was  made  by  earlier  investigators  (Stephens  and 
Myers  *)  that  red  blood-cells  which  are  soluble  in  weak  solutions  of 
poison  may  be  insoluble  hi  stronger  solutions;  and  the  same  observa- 
tion was  made  by  us  on  rabbit  blood.  This  phenomenon  is  entirely 
irreconcilable  with  the  assumption  of  a  preformed  poison,  for,  ceteris 
paribus,  the  action  of  this  should  increase  with  the  dose.  This  inhibi- 
tion in  consequence  of  large  doses  of  poison  cannot  be  harmonized 
with  the  toxin  theory 

On  the  contrary  it  indicates  that  we  are  here  dealing  with  a  phe- 
nomenon whose  significance  was  first  pointed  out  by  M.  Neisser  and 
Wechsberg,2  which  consists  hi  this,  that  the  bactericidal  action  of  an 
immune  serum,  provided  the  amount  of  complement  remains  the 
same,  is  inhibited  by  an  excess  of  amboceptor. 

If  we  assume  that  the  red  blood-cells  in  themselves  possess  a 
complement  fitting  the  amboceptor  of  the  cobra  poison,  an  "endo- 
complement/'  we  see  at  once  that  small  amounts  of  amboceptor  effect 
solution,  while  with  large  doses  no  solution  occurs  owing  ta  diversion  of 
the  complement  by  the  amboceptors.  This  diversion  is  due  to  the  mass 
action  of  the  amboceptors  present  in  the  fluid.  This  view  is  easily 
supported  experimentally.  If  blood-cells  are  treated  with  a  solution 

1See  also  page  443. 

2  Journal  of  Pathology  and  Bacteriology,  Vol.  V,  1898. 

3  Munch,  med.  Wochenschj.  1901,  No.  18.    See  also  page  120. 


296 


COLLECTED  STUDIES  IN  IMMUNITY. 


of  very  strong  snake  poison,  they  will  not  be  dissolved.  The  mixture 
is  now  centrifuged  and  the  sediment  washed  with  salt  solution.  No 
solution  takes  place;  as  soon  as  fitting  complement  is  added,  however, 
solution  ensues  very  promptly.  This  shows  that  by  the  treatment  with 
the  poison  the  complement  contained  in  the  red  blood-cells  has  been 
abstracted.  The  following  diagram  will  make  this  clear. 


I.  Blood-cell  with  receptor  r  and  endocomplement  e. 

II.  Blood-cell  after  treatment  with  a  large  amount  of  cobra  poison.  The 
cobra  amboceptor  c  has  been  anchored  by  the  blood-cell  receptor.  The 
endocomplement  has  been  abstracted  from  the  cells  by  the  large  excess  of 
free  amboceptor. 

III.  Blood-cell  of  stage  II  after  the  addition  of  complement  or  endocomplement  e. 
The  added  endocomplement  has  combined  with  the  cobra  amboceptor  c 
and  can  now  effect  solution. 


The  following  experiment  may  serve  as  an  illustration.      (See 
Table  III.) 

TABLE  III. 


1  cc.  5%  Rabbit  Blood  +  1  cc.  5%  Cobra  Poison, 
Kept   at  37°  for  Two  Hours,  Centrifuged  and 
Washed..   Sediments  + 

Controls  Native 
Rabbit  Blood 
+ 
0.15  cc.  Guinea- 
pig  Serum  or 
0.5  cc.  Guinea- 
pig  Endocomple- 
ment. 

a 

0.85% 
NaCl  Solution. 

b 

0.15  cc.  Guinea- 
pig  Serum. 

c 
0.5  cc.  Guinea- 
pig  Endocom- 
plement (i). 

Solution  effected 

0 

complete 

complete 

0 

The  correctness  of  this  view  can  readily  be  shown  in  another 
way  If  the  blood-cells  actually  do  contain  an  endocomplement,  it 
must  be  possible  to  demonstrate  this  by  dissolving  the  blood-cells  in 
water  and  finding  that  these  dissolved  cells  are  capable  of  acting  as 
complement  to  cobra  poison  for  such  blood-cells  as  are  incapable  of 
being  dissolved  by  cobra  poison  alone. 


THE  MODE  OF  ACTION  OF  COBRA  VENOM. 


297 


As  a  matter  of  fact  we  have  succeeded  in  a  large  number  of  cases 
in  causing  the  solution  of  such  cells  by  the  addition  of  laky  solutions 
of  endocomplement.1  The  amount  of  endocomplement  contained  in 
blood-cells  varies;  that  of  human  and  guinea-pig  blood  appears 
to  be  the  highest  and  also  fairly  constant. 

The  following  table  shows  the  combinations  in  which,  according 
to  our  experiments,  cobra  poison  causes  solution  (  +  )  of  blood-cells 
which  are  not  dissolved  by  cobra  poison  alone  (see  Table  IV). 

TABLE  IV. 


Endocomplement  of 

Species  of  Blood. 

Ox. 

Goa 

Sheep. 

Rabbit 

+ 
+ 
+ 
+ 

_2 

+ 
2 

+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 

Man               

Doe 

Guinea-pig  

Goat   

Ox 

Sheep 

It  is  in  place  here  to  mention  another  fact.  The  deflection  of  the 
endocomplement  by  large  quantities  of  poison  described  in  the  case 
of  blood-cells  vulnerable  to  cobra  poison  succeeds  equally  well  if 
the  experiment  is  made  with  blood-cells  insensitive  to  cobra  poison 
alone  (ox  blood)  and  if  dissolved  endocomplements  (guinea-pig)  are 
used  for  activation.  There  is  no  doubt  therefore  that  the  blood-cells 
themselves  contain  complement-like  substances,  endocomplements. 

So  far  as  the  behavior  of  these  endocomplements  toward  thermic 
influences  is  concerned,  they  are  seen  to  be  somewhat  more  resistant 
in  general  than  are  the  complements  contained  in  the  serum,  for  it 
requires  half  an  hour's  heating  to  62°  C.  to  inactivate  them  (see  Table 
V).  In  the  light  of  our  present  knowledge,  however,  we  probably 
cannot  deny  the  complement  character  of  these  substances  merely 

1  As  a  rule  these  endocomplement  solutions  were  prepared  by  twice  washing 
and  centrifuging  a  certain  quantity  of  full  blood,  and  then  filling  the  sediment 
up  to  a  certain  volume.     Either  the  original  volume  or  a  greater  or  less  dilu- 
tion was  made  up  depending  on  circumstances.      They  were  then  salted  to 
contain  0.85%  NaCl.      We  have  designated  these  dilutions  as  J,  $,  -fa,  etc.,  endo- 
complement. 

2  Even  in  these  cases  we  noticed  an  activation  with  certain  specimens  of 
blood. 


298  COLLECTED  STUDIES  IX  IMMUNITY. 

because  of  this  thermostability,  especially  since  we  know  that 
Ehrlich  and  Morgenroth 1  have  described  a  partial  complement 
in  goat  serum  which  was  much  more  thermostable.  According  to 
some  unpublished  studies  by  Shiga  such  thermostable  complements 
seem  to  take  part  in  the  bacteriolysis  of  anthrax  bacilli  by  rabbit  serum. 
The  active  group  of  coagulins  and  agglutinins,  which,  according  to 
Ehrlich,  is  analogous  to  the  zymotoxic  group  of  complements,  is  still 
more  thermostable,2  for  inactivation  takes  place  only  between  70  and 
75°  C. 

From  all  this  it  follows  that  we  must  assume  the  blood-cells  which 
are  sensitive  to  the  above  poison,  to  be  supplied  both  with  receptors 
and  complements.  Through  the  intervention  of  the  amboceptors 
added,  the  discoplasma  enters  into  that  intimate  combination  with 
the  complement  which  is  necessary  in  order  that  the  latter  may  act. 

We  should  like  to  add  a  few  explanatory  remarks  to  these  state- 
ments, and  shall  begin  with  the  conception  of  complements  as  endocom- 
plements.  One  could,  for  example,  assume  that  the  endocomple- 
ments  are  derived  not  from  blood-cells  themselves  but  from  the  serum 
still  adherent  to  these.  However,  we  believe  that  the  repeated  wash- 
ing and  centrifuging  has  completely  freed  the  red  blood-cells  from 
serum.  Guinea-pig  blood-cells  were  washed  and  centrifuged  eight 
times,  yet  even  after  that  the  dissolved  blood-cells  manifested  the 
complement  action.  This  excludes  the  possibility  of  the  action  being 
due  to  adherent  serum.  Another  thing  which  speaks  against  this  is 
the  fact,  now  and  then  observed  by  us  (mostly,  to  be  sure,  merely 
indicated)  that  the  last  decantations  activated  more  strongly  even 
than  the  first.  If  the  washing  removed  adherent  serum  constituents, 
the  first  washings  should  contain  more  than  the  later  ones.  As 
a  matter  of  fact  just  the  reverse  was  found  to  be  the  case;  which 
indicates  that  we  are  dealing  with  an  extraction  phenomenon. 

In  one  case  we  even  succeeded  in  entirely  removing  the  endo- 
complement  by  means  of  salt  solution.  This  was  a  suspension 
(5%  in  0.85%  salt  solution)  of  rabbit  blood,  which  is  dissolved 
by  cobra  poison.  This  suspension  was  kept  in  a  refrigerator  for 
twenty-four  hours  and  then  centrifuged,  when  it  was  found  that 
the  sedimented  blood-cells  suspended  in  fresh  salt  solution  were  no 

1  See  pages  11  et  seq. 

2  See  Bail,  Archiv  fur  Hygiene,  Vol.  XLII,  1902;   also  Eisenberg  and  Volk, 
Zeitschr.  f.  Hygiene,  Vol.  XXXIV,  1902. 


THE  MODE  OF  ACTION  OF  COBRA  VENOM. 

TABLE   V. 
IN  ALL  CASES  0.02  cc.  1%  COBRA  POISON. 


299 


Amount 
of  the 

1  cc.  5%  Ox  Blood  +  Guinea-pig  Blood  Endocomplement  (1/20). 

Endocom- 

plement 

6 

(1/20). 

cc. 

Normal. 

Heated  to  62°  for  %  Hour. 

1.0 

complete 

0 

0.75 

0 

0.5 

(  i 

0 

0.25 

trace 

0 

0.1 

0 

0 

B. 


1  cc.  5%  Goat  Blood  +  Guinea-pig  Blood  Endocomplement  (1/10). 


Endocom- 
plement 

Heated  Half  an  Hour  to 

(1/10). 

i 

a 

6 

c 

d 

cc. 

Normal. 

56°  C. 

60°  C. 

62°  C. 

1.0 
0.5 

complete 
moderate 

strong 
little 

trace 

i  i 

0 
0 

0.25 

little 

trace 

0 

0 

0.1 

faint  trace 

0 

0 

0 

1  cc.  5%  Sheep  Blood  +  Guinea-pig  Endocomplement  (1/10). 


Endocom- 
plement 

Heated  Half  Hour  to 

(1/10). 

a 

b 

c 

d 

cc. 

Normal. 

56°  C. 

60°  C. 

62°  C. 

1.0 

almost  complete 

moderate 

trace 

0 

0.5 

little 

trace 

0 

0 

0.25 

trace 

1  1 

0 

0 

0.1 

0 

0 

0 

0 

longer  dissolved  by  the  cobra  poison,  or  were  only  very  slightly 
dissolved.  If  our  view  was  correct,  the  endocomplements  would  now 
be  found  in  the  decanted  fluid.  This  proved  to  be  the  case,  for  the 
addition  of  suitable  amounts  of  this  fluid  sufficed  to  cause  solution 
of  the  blood-cells  which  were  insoluble  in  cobra  poison  alone.  We 


300  COLLECTED  STUDIES  IN  IMMUNITY. 

were  unable  to  obtain  a  like  result  in  two  similar  cases.  Evidently 
slight  variations  in  the  experiment  and  possibly  also  minute  changes, 
and  impurities  leading  perhaps  to  certain  ion  actions,  play  a  role  which 
it  is  difficult  to  control.  We  were  not  interested  enough  to  follow 
up  these  relations;  but  we  believe  that  had  we  done  so  we  could  have 
made  the  conditions  more  favorable  for  washing  out  the  endocomple- 
ments.  We  merely  mention  this  because  Flexner  and  Noguchi  state 
that  in  their  experiments  after  repeated  washings  of  the  blood-cells 
all  of  these  were  found  insoluble  in  cobra  poison  alone. 

These  authors  did  most  of  their  work  with  snake  poisons  differ- 
ent from  ours  (Crotalus  adamanteus,  Ancistrodon  contortrix,  etc.). 
How  far  this  fact  is  responsible  for  the  divergence  cannot  here  be 
decided,  nor  whether  the  escape  of  the  endocomplements  was  favored 
by  other  conditions  in  the  experiments.1 

That  the  endocomplements  cannot  be  derived  from  the  serum 
is  also  shown  by  the  observation  frequently  made  by  us  that  the  serum 
of  several  species  of  blood,  whose  blood-cells  exhibit  a  plentiful  supply 
of  endocomplement,  does  not  possess  the  slightest  activating  power, 
but  that,  on  the  contrary  (as  in  the  case  of  rabbit  serum) ,  it  sometimes 
hinders  haemolysis  of  the  homologous  blood-cells  by  snake  poison. 

So  far  as  the  condition  is  concerned  in  which  the  endocomple- 
ments exist,  we  must  assume,  in  those  cases  in  which  the  blood- 
cells  are  directly  soluble,  that  the  endocomplement  is  contained 
free  in  the  blood-cells.  In  those  blood-cells,  which  are  primarily 
insoluble,  it  will  either  be  absent  or  be  present  in  a  latent  form. 
We  believe  the  endocomplements  are  absent  in  the  goat,  for  in  no 
case  were  the  dissolved  goat  blood-cells  able  to  activate  cobra  venom 
for  goat  blood.  On  the  other  hand,  ox  blood  is  sensitized  for  cobra 
venom  by  dissolved  ox  blood-cells,  so  that  we  shall  have  to  assume 
that  ox  blood  does  not  contain  endocomplements  in  available  form 
and  that  these  endocomplements  are  changed  into  an  active  form 
when  the  cells  are  dissolved. 

We  shall  reserve  for  subsequent  study  the  question  as  to  whether 
the  endocomplements  are  of  simple  constitution  or  complex. 

Attention  is  called  to  the  fact  that  the  existence  of  endocom- 
plements furnishes  another  objection  to  Bordet's  view  that  the 

1  We  shall  merely  say  that  Daboia  poison,  which  through  Lamb's  pretty 
experiments  has  been  shown  to  differ  from  cobra  poison,  does  not  dissolve  rabbit 
blood. 


THE  MODE  OF  ACTION  OF  COBRA  VEXOM.       301 

amboceptor  is  only  a  key  which  makes  possible  the  entrance  of  the 
complement  into  cell.  For  in  these  cases  the  complements  which 
are  able  to  destroy  the  blood-cell  are  already  present  within  the 
same  before  the  amboceptor  is  anchored,  and  yet  the  blood-cell  is 
in  no  way  injured.  The  injury  takes  place  only  when  a  particular 
organic  relation  has  been  effected  between  complement  and  protoplasm 
by  means  of  the  amboceptor. 

Finally,  the  demonstration  that  the  red  blood-cells  contain  com- 
plementing substances  is  exceedingly  important  in  other  directions. 
The  French  school  in  particular  was  inclined  to  refer  the  source  of 
the  complements  exclusively  to  the  leucocytes.  We  now  see  that 
the  red  blood-cells,  heretofore  considered  merely  as  concerned  with 
the  oxygen  exchange,  are  also  carriers  of  special  complement-like 
substances.  This  confirms  the  view  expressed  by  Ehrlich 1  in  his 
"Schlussbetrachtungen,"  namely,  that  "the  red  blood-discs  also 
exercise  other  functions  hitherto  overlooked."  "The  red  blood-cells 
serve  as  storage  centres  in  the  sense  that  they  temporarily  take  up 
into  themselves  substances  characterized  by  the  presence  of  hapto- 
phore  groups  and  derived  from  the  internal  metabolism  or  from  the 
food." 

III.  Cobra  Venom  and  Lecithin. 

Having  demonstrated  that  the  amboceptor  of  snake  poison  can 
be  complemented  by  easily  destructible  complements  which  may  be 
present  either  in  the  serum  or  in  the  red  blood-cells,  we  go  on  to  a 
series  of  other  phenomena  in  which  activation  is  effected  by  more 
stable  substances  which  are  in  no  way  related  to  the  complements. 
Calmette,2  in  following  up  the  work  of  Flexner  and  Noguchi,  found 
that  certain  normal  sera  when  heated  to  62°  C.  became  much  better 
able,  in  conjunction  with  cobra  poison,  to  cause  haemolysis  of  the 
washed  blood-cells.  In  fact  it  was  found  that  fresh  sera,  added  hi 
large  excess,  can  retard  or  even  inhibit  haemolysis,  while  these  same 
sera  when  heated  cause  immediate  solution  of  the  blood-cells  in  the 
presence  of  cobra  poison.  From  this  Calmette  concludes  that  such  a 
blood  serum  must  contain  a  natural  antihsemolysin  which  can  pro- 
tect the  red  blood-cells  up  to  a  certain  point  against  solution  by  the 

1  In  Nothnagel's  Specielle  Pathologic  und  Therapie,  Vol.  8.     Vienna,  1901. 

2  I.e. 


302  COLLECTED  STUDIES  IN  IMMUNITY. 

snake  venom.  This  antihsemolysin  is  thermolabile,  being  destroyed 
by  temperatures  over  56°  C.  The  other  (the  activating)  constituent 
of  the  serum  on  the  contrary  is  thermostable,  since  it  does  not  lose 
its  activity  even  by  heating  to  80°  C.  Calmette  therefore  assumes 
that  the  alexin  (our  complement)  takes  no  part  in  the  activation,  but 
that  a  particularly  thermostable  "substance  sensibilatrice "  is  con- 
tained in  the  serum  besides  the  thermolabile  antihcemolysin.  By  the 
term  "substance  sensibilatrice/'  as  used  in  French  terminology,  is 
meant  the  body  which  we  term  "amboceptor."  The  amboceptor 
capable  of  being  anchored  is  supposed  to  render  the  red  blood-cells 
sensitive  to  the  attack  of  the  alexin  (complement).  It  is  hard  to 
see  just  how  Calmette  conceives  this  entire  process.  As  we  already 
know  from  the  researches  of  Flexner  and  Noguchi  snake  venom  is 
capable  of  being  anchored,  and  from  all  of  its  properties  is  therefore 
surely  a  substance  sensibilatrice  (amboceptor) .  If  then  the  substance 
supposed  by  Calmette  were  also  a  sensitizer,  we  should  have  before 
us  something  absolutely  unique,  namely,  the  combined  action  of  two 
sensitizers.  Unfortunately  Calmette  has  undertaken  no  combining 
experiments  and  therefore  has  furnished  no  proof  for  his  view.  Our 
own  experiments,  however,  speak  against  this  assumption. 

In  our  opinion  the  main  reasons  which  led  Calmette  to  conclude 
that  complements  play  no  role  in  the  haemolysis  by  means  of  cobra 
venom  are : 

1.  That  he  overlooked  the  endocomplements. 

2.  That  he  employed  too  schematic  a  manner  of  activation,  namely ,. 
usually  only  at  62°  C. 

We  have  convinced  ourselves  that  in  suitable  cases  (see  Table 
VII,  case  IV)  a  blood  serum,  e.g.  ox  serum,  when  fresh,  dissolves 
the  red  blood-cells.  If  this  is  inactivated  by  heating  to  56°  C.,  the 
action  will  be  found  to  be  completely  inhibited,  or  almost  so.  This 
same  serum,  however,  when  heated  to  65°  C.  or  higher  is  again  able 
to  effect  haemolysis.  The  serum  heated  in  this  manner  possesses  a 
stronger  solvent  power  than  the  fresh  serum,  for  even  fractional 
parts  of  the  complete  solvent  dose  of  fresh  serum  suffice  to  cause 
full  solution  (see  Table  VI). 

This  experiment  was  repeated  many  times  and  proves  that  in 
this  case  two  entirely  different  kinds  of  activations  occur,  namely, 

1.  Activation  by  means  of  complements. 

2.  Activation  by  means  of  substances  which  become  manifest  only 
through  heating. 


THE  MODE  OF  ACTION  OF  COBRA  VENOM. 


303 


TABLE   VI. 
1  cc.  5%  HORSE  BLOOD  4- Ox  SERUM. 


1 

I. 

II. 

0.02  cc.  1%  Cobra  Poison  +  Ox  Serum,  1/10  Dilution. 

Amount 
of  the 
Ox  Serum 

Ox  Serum  Alone. 

Heated  for  One  Half-hour  to 

(1/10). 

a 

b 

c 

cc. 

Normal. 

56°  C. 

65°  C. 

0.5 
0.35 

faint  trace 
0 

complete 
almost  complete 

faint  trace 

complete 

0.25 

0 

strong 

0 

i  c 

0.15 

0 

little 

0 

little 

0.1 

0 

trace 

0 

trace 

It  seemed  to  us  that  it  was  of  the  highest  importance  to  gain 
a  further  insight  into  these  thermostable  activating  substances.  To 
begin,  we  found  that  the  substance  is  far  more  stable  than  Calmette 
assumed,  for  activation  is  effected  even  by  sera  which  have  been 
cooked  for  hours.  Thereupon  we  investigated  a  number  of  sera  in 
respect  to  their  activating  power  and  obtained  results  that  were 
little  less  than  confusing.  We  found  sera  which  activated  not  only 
in  the  fresh  state  but  also  after  heating  to  56°  C.  and  100°  C.  (No.  I 
of  Table  VII).  Other  sera  did  not  activate  either  when  fresh  or 
after  heating  to  56°  C. ;  they  did  activate,  however,  when  they  were 
heated  to  65°  and  100°  C.  (No.  II  of  Table  VII).  As  a  rule  in  these 
cases  the  serum  heated  to  100°  C.  proved  more  powerful  than  that 
heated  to  65°  C.  A  third  class  of  sera  was  found  which  did  not  activate 
when  fresh,  but  activated  when  heated  to  56°  C.  or  higher  (No.  Ill  of 
Table  VII).  Finally,  there  is  the  type  already  mentioned,  namely,  a 
serum  which  activates  when  fresh,  is  made  inactive  by  heating  to 
56°  C.  and  again  made  active  by  heating  to  65°  (No:  IV  of  Table  VII). 
We  have  also  observed  sera  which  activate  only  when  fresh  and  do  not 
again  acquire  this  property  when  heated  to  a  greater  or  less  degree 
(No.  V  of  Table  VIII).  We  see  therefore  that  we  are  dealing  with 
five  different  combinations,1  as  is  shown  in  Table  VII. 

1  Naturally  in  the  case  of  such  bloods  as  rabbit  blood,  which  are  dissolved 
by  cobra  poison  alone,  only  such  amounts  of  poison  have  been  used  which  by 
themselves  are  not  active,  but  which  cause  haemolysis  when  they  are  combined 
with  suitable  reinforcing  agents  (complements,  etc.). 


304 


COLLECTED  STUDIES  IN  IMMUNITY. 
TABLE  VII. 


Activating  Power  of  the  Serum. 

Combinations. 

a 

6 

Heated  to 

Serum. 

Blood-cell. 

Normal. 

56°  C. 

65°  or  100°  C. 

f 

horse 

ox 

horse 

goat  * 

I 

+ 

+ 

+          i 

horse 

horse 

man 

man 

I 

rabbbit 

ox 

man 

goat* 

II 

0 

0 

+  { 

man 
sheep 
rabbit 

ox 
sheep  * 
goat  * 

III 

0 

+ 

+  ( 

ox 
sheep 

ox 
ox 

guinea-pig 

ox 

IV 

+ 

0 

+ 

ox 

horse 

•  V 

+ 

0 

0 

guinea-pig 
guinea-pig 

sheep  * 
rabbit 

*  Only  slight  solution. 

These  contradictory  results  are  not  to  be  harmonized  with  Cal- 
mette's  conception  of  a  definite  antibody  which  is  destroyed  at  56°  C. 
One  would  have  to  assume  that  this  normal  antihaemolysin  were 
lacking  in  horse  serum,  for  as  a  rule  this  does  not  become  more  strongly 
hsemolytic  by  heating  to  56°  C.  On  the  other  hand  in  the  case  of  a 
serum  like  No.  II,  which  has  no  activating  properties  even  when 
heated  to  56°  C.,  it  would  be  necessary  to  believe  that  the  activator 
is  entirely  absent.  The  conditions  are  still  more  complicated  by 
the  fact  that  one  and  the  same  serum  can  behave  differently  toward 
.various  species  of  blood.  Thus  a  horse  serum  heated  to  100°  will 
activate  cobra  venom  for  ox  blood  in  high  dilutions  (0.02  complete), 
whereas  even  in  large  amounts  it  dissolves  goat  blood  only  in  com- 
paratively slight  degree  (0.35  cc.  moderate  solution).  In  this  case, 
then,  the  activator  present  is  in  the  main  one  for  ox  blood,  not  for 
goat  blood. 

Believing  that  an  insight  into  the  nature  of  this  maze  of  facts 
could  be  gained  only  by  a  thorough  chemical  analysis,  we  sought  to 
isolate  the  thermostable  activating  substance.  First  we  succeeded 
in  proving  that  when  serum  is  precipitated  with  8  to  10  volumes  of 
alcohol,  the  activating  substance  passes  into  the  alcohol,  while  the 
inhibiting  substance  is  contained  in  the  precipitate.  For  if  the 


THE  MODE  OF  ACTION  OF  COBRA  VENOM.       305 

alcoholic  extract  is  evaporated  in  vacuo  and  the  residue  dissolved  in 
an  amount  of  0.85%  salt  solution  equal  to  the  original  amount  of 
serum,  a  strong  activating  fluid  is  obtained.  An  alcoholic  extract  of 
horse  serum,  when  treated  hi  this  way,  hi  contrast  to  the  native 
horse  serum  heated  to  100°  C.,  dissolves  goat  blood  to  a  high  degree 
(0.1  cc.  dissolves  completely).  The  alcohol  precipitate  must  there- 
fore have  contained  a  substance  which  inhibits  the  action  of  the 
activator,  and  we  were  actually  able  to  demonstrate  the  existence  of 
this  inhibiting  substance.  If  the  precipitate  is  dissolved  in  salt  water, 
a  fluid  is  obtained  which  inhibits  the  haemolysis  of  goat  blood  by 
cobra  venom  and  the  activator  derived  from  the  alcoholic  extract  of 
horse  serum.  In  larger,  though  unequal,  doses  it  protects  ox  blood 
against  solution  by  cobra  venom  and  the  activator.  Before  studying 
the  nature  of  the  inhibition  effected  by  the  albuminous  precipitate 
we  shall  try  to  discover  the  nature  of  the  activator.  As  already  said, 
the  residue  obtained  on  evaporating  the  alcoholic  extract  was  dissolved 
in  salt  water.  On  shaking  this  solution  with  ether,  we  found  that 
the  ether  had  taken  up  all  of  the  activating  substance.  This  proved 
that  the  activator  is  a  substance  soluble  both  in  alcohol  and  ether, 
and  one  which  has  a  wide  distribution  in  the  sera  of  animals.  Constit- 
uents of  the  blood  serum  which  are  soluble  hi  ether  have  long  been 
known  to  us.  Those  mainly  to  be  considered  are  cholesterin,  lecithin, 
fats  and  fatty  acids.  After  several  negative  trials  with  cholesterin 
we  found  that  lecithin  possesses  the  properties  of  the  activator,  since 
all  blood-cells  are  rapidly  dissolved  when  cobra  venom  and  lecithin 
are  allowed  to  act  on  them  simultaneously.  Not  only  blood-cells  which 
are  insoluble  in  cobra  venom  alone,  such  as  goat  blood-cells,  but  also 
those  which  are  deprived  of  endocomplements  when  treated  with 
strong  solutions  of  poison  (see  §  II,  Endocomplements)  are  promptly 
dissolved  by  the  lecithin.  Our  solution  of  lecithin  l  was  made  in  the 


1  The  lecithin  employed  by  us  was  derived  from  yolk  of  egg  and  obtained 
from  E.  Merck,  Darmstadt.  It  was  a  neutral  mass  of  salve-like  consistency, 
which  was  entirely  precipitated  from  its  ethereal  solution  by  aceton  (Altnlann- 
Henriquez).  Even  when  thus  purified  it  manifested  the  activating  power 
unchanged.  We  reserve  for  further  study  our  experiments  with  the  pure  lecithin 
prepared  after  the  method  of  P.  Bergell  (Ber.  der  deutsch.  chem.  Gesellschaft, 
Jahrg.  33,  1900,  page  2584)  and  with  the  homologues  of  this  body.  A  specimen 
of  lecithin  obtained  from  J.  D.  Riedel,  Berlin,  corresponded  exactly  in  its  activity 
to  Merck's  lecithin.  Cerebrin  and  Protagon,  obtained  through  the  courtesy  of 
Prof.  Kossel  of  Heidelberg,  possess  no  activating  power. 


306 


COLLECTED  STUDIES  IN  IMMUNITY. 


purest  methyl  alcohol,  for,  as  we  know  from  special  experiments,  this 
does  not  injure  the  red  blood-cells  even  in  concentration  up  to  9  or 
10%.  A  1%  stock  solution  was  diluted  with  0.85%  salt  solution,  and 
it  was  found  that  0.0025  cc.  to  0.0035  cc.  of  the  1%  solution  (i.e. 
0.000025  g.  lecithin)  were  sufficient  to  completely  dissolve  1  cc.  5% 
ox  or  goat  blood  on  the  addition  of  suitable  amounts  of  cobra  poison. 
(See  Table  VIII). 

TABLE  VIII. 


Amount  of  the 
1%  Lecithin. 

0.002  cc.  1%  Cobra  Poison. 

Ox  Blood. 

Goat  Blood. 

0.005 

0.0035 
0.0025 
0.0015 
0.001 
0.00075 

complete 

it 

(  t 

almost  complete 
little 
0 

complete 

1  1 

moderate 
trace 
0 
0 

In  what  way  now  are  we  to  picture  the  action  of  this  lecithin? 
We  know  that  lecithin  is  able  to  combine  with  albuminous  bodies, 
sugars  (Henriquez  and  Bing),  etc.  A  threefold  question  had  to  be 
decided.  First,  whether  cobra  venom  unites  with  lecithin  after  the 
fashion  of  an  amboceptor;  second,  whether  perhaps  the  snake  venom 
had  made  the  blood-cells  sensitive  to  lecithin;  or  third,  whether 
the  reverse  holds  true. 

A  preliminary  test  was  made  to  see  whether  lecithin  and  snake 
poison  combine  with  one  another.  The  method  of  making  this 
experiment  is  relatively  simple.  Lecithin  can  easily  be  shaken  out 
of  its  solution  in  salt  water  by  means  of  ether.  As  the  following 
experiment  will  show,  lecithin  passes  into  the  ether  in  great  abundance, 
but  not  completely.  This  behavior  corresponds  to  a  general  phe- 
nomenon which  is  the  expression  of  the  "loi  de  partage."  If,  how- 
ever, to  the  same  amount  of  lecithin  a  suitable  quantity  of  snake 
venom  is  added,  it  is  found  that  but  very  little  passes  into  the  ether 
on  shaking  the  ether  with  the  mixture.  Two  portions  each  of  10  cc. 
were  thus  shaken  out  with  ether:  A,  containing  2  cc.  of  a  certain 
lecithin  solution:  B,  containing  besides  this  1  cc.  of  a  1%  solution 
of  cobra  venom.  Previous  to  this  both  solutions  were  kept  at  37°  C. 
for  half  an  hour.  The  ethereal  extract  was  evaporated  and  the 
residue  taken  up  in  10  cc.  0.85%  salt  solution  The  action,  on  ox 


THE  MODE  OF  ACTION  OF  COBRA  VENOM. 


307 


blood  4- cobra  venom,  of  the  ethereal  extract  residues  on  the  one  hand, 
and  of  the  solutions  which  had  been  shaken  out  on  the  other,  is 
shown  in  Table  IX. 

TABLE  IX. 

Complete  solvent  dose  of  lecithin  (stock  solution)  with  0.1  cc.  of  0.1%  cobra 
venom  =0.005  cc.  (corresponding  to  0.025  cc.  of  the  shaken-out  solution). 


1  cc.  5%  Ox  Blood  +0.1  cc.  0,1%  Cobra  Poison. 

Amount 
of  A  or 
of  B. 

A.  Lecithin  Only. 

B.  Lecithin  +  Cobra  Poison. 

I. 

II. 

I. 

II. 

Ether  Extract. 

Aqueous  Portion. 

Ether  Extract. 

Aqueous  Portion. 

1.0 

complete  solution 

complete  solution 

complete  solution 

complete  solution 

0.5 

if           it 

ti           it 

moderate 

t  i           (  t 

0.25 

it           a 

tt           1  1 

0 

t  <           tt 

0.1 

11           n 

0 

— 

it            n 

0.05 

i  (           1  1 

— 

— 

it           1  1 

0.025 

trace 

— 

— 

a            1  1 

0.015 

0 

~ 

— 

0 

It  can  be  seen  from  the  table  that  on  the  addition  of  snake 
poison  to  the  same  lecithin  solution  only  -£$  part  of  that  amount 
of  lecithin  passed  into  the  ether  which  passes  into  ether  when  a 
pure  lecithin  solution  is  shaken  out.  The  cobra  venom  had  there- 
fore bound  the  lecithin. 

The  next  question  to  determine  was  how  the  red  blood-cells 
behaved  toward  cobra  venom  and  lecithin  alone  and  toward  mixtures 
of  these  substances.  In  order  to  retard  the  course  of  the  reactions  as 
much  as  possible  and  to  secure  a  better  view  of  the  processes  we 
sought  to  create  such  retarding  conditions  by  making  the  test 
with  dilute  solutions  and  at  0°  C.  This  necessitated  a  preliminary 
quantitative  determination  of  the  effect  of  each  factor  separately. 
Corresponding  to  the  slight  affinity  of  the  cobra  amboceptor  for  the 
red  blood-cells,  it  was  found  that  with  suitable  conditions  (2  hours  at 
0°  in  dilute  solutions  of  the  poison)  the  amboceptor  is  not  anchored; 
neither  is  lecithin  by  itself  bound  by  the  blood-cells.  On  the  other 
hand  blood-cells  to  which  cobra  venom  +  lecithin  were  added  in 
suitable  quantities  were  rapidly  dissolved  even  at  0°  C.  Both  com- 
ponents must  therefore  have  been  bound.  The  following  table 
(Table  X)  illustrates  this  behavior. 


308 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE  X. 

Complete  solvent  dose  of  cobra  venom  (0.1%)  in  the  presence  of  0.01  cc.  lecithin 
=  0.005  cc.  Complete  solvent  dose  of  lecithin  in  the  presence  of  0.1  cobra 
venom  (0.1%)  =0.005  cc. 


Amount  of 
Cobra  Venom 
Added 
(0.1%). 
cc. 

1  cc.  5%  Ox  Blood  +  Decreasing  Amounts  of  Cobra  Venom  Kept  Two  Hours 
at  0°,  then  Centrifuged  and  Washed.     Thereupon  0.01  Lecithin  Solution 
Added  to 

I. 

The  Sediments. 

II. 
To  the  Decanted  Fluid  which  had  been 
Added  to  Native  Ox  Blood. 

0.01 
0.05 
0.025 
0.01 
0.005 
0.0025 

faint  trace  solution 
0 
0 
0 
0 
0 

complete  solution 

tt              « 

ti              it 
((              ii 

almost  complete 
0 

B. 


1  cc.  5%  Ox  Blood  +  Decreasing  Amounts  of  Lecithin  Kept  at  0°  C.  for  Two 


Amount  of 
the  Lecithin 

jtiours,   tnen  i_/entniugea  ana  was 
(0.01%)  Added  to 

Solution 
Added. 

QC. 

I. 
The  Sediments. 

II. 
To  the  Decanted  Fluid  which  had 
Added  to  Native  Ox  Blood. 

been 

0.075 
0.05 
0.025 
0.01 
0.0075 
0.005 

trace  solution 
tt           < 

(i           t 

ft                     C 

1  1           f 

it           t 

complete  solution 

«              1  1 

(C                           tt 

I  (                     It 
It                 tt 

0 

C. 


Amount  of 
Cobra  Venom 
Added 
(0.1%). 
cc. 

1  cc.  5%  Ox  Blood  +  0.025  Lecithin  Solution  +  Decreasing  Amounts  of  Cobra 
Venom,  Two  Hours  at  0°  C. 

I. 

Degree  of  Solution 
Effected. 

II. 

Specimens  not  Dissolved  are  Centrifuged,  the 
Sediments  Washed. 

a 
Sediments  Suspended  in 
Salt  Solution. 
(+0.01  cc.  Lecithin). 

b 

Decanted  Fluids  Poured 
over  Native  Ox  Blood. 

0.1 
0.05 
0.025 
0.01 
0.005 
0.0025 
0.001 
0 

complete 

tt 
1  1 

faint  trace 
0 
0 
0 

0 
0 
0 
0 

complete 
moderate 
0 
0 

THE  MODE  OF  ACTION  OF  COBRA  VENOM.       309 

These  results  can  be  explained  only  by  assuming  that  lecithin 
and  cobra  amboceptor  have  combined  to  form  what  may  be  termed 
the  "lecithin"  of  cobra  poison,  and  that  the  affinity  of  the  cobra 
amboceptors  cytophile  group  is  thereby  increased.  According  to  this 
the  union  with  the  lecithin  causes  the  cobra  poison  to  be  more 
rapidly  anchored  than  the  cobra  amboceptor  alone.  The  increase  of 
the  cytophile  groups  affinity  through  the  occupation  of  another 
grbup  is  perfectly  conceivable  chemically.  An  analogy  frequently 
met  with  is  the  fact  that  the  anchoring  of  the  hsemotytic  serum 
amboceptors  by  the  blood-cells  usually  causes  an  increase  in  the 
affinity  of  the  complementophile  group.  Ehrlich  and  Sachs  l  have 
shown  that  the  occupation  of  the  complementophile  group  of  serum 
amboceptors  can  cause  an  increase  of  the  cytophile  group's  affinity, 
such  as  is  presented  in  this  case. 

We  therefore  assume  that  the  lecithin  acts  as  a  kind  of  comple- 
ment since  it  is  anchored  by  certain  definite  groupings  of  the  poison 
molecule.  In  this  way  a  poisonous  double  combination  is  formed 
of  which  perhaps  the  cholin  residue  constitutes  the  toxophore  group. 

There  is  another  fact  which  supports  the  view  here  presented, 
namely,  that  the  lecithin  amboceptors  effect  solution  of  the  red  blood- 
cells  even  at  0°  C.,  whereas  the  thermolabile  complements  of  blood 
serum  are  anchored  only  at  higher  temperatures.  Corresponding  to  the 
views  formulated  by  Ehrlich  and  Marshall 2  for  the  amboceptors 
(polyceptors)  of  blood  serum,  we  must  therefore  assume  that  the 
snake  venom  amboceptor  in  addition  to  its  cytophile  group  possesses 
at  least  two  haptophore  groups,  of  which  one  as  usual  is  able  to  bind 
complements,  the  other  to  bind  lecithin.  Each  of  these  combina- 
tions by  itself  is  dominant,  i.e.,  sufficient  to  effect  solution  of  the  blood- 
cells.  It  is  very  probable  that  occupation  of  both  groups  increases 
the  solvent  effect. 

The  following  experiment  furnishes  additional .  proof  that  the 
phenomena  observed  cannot  be  regarded  in  the  light  of  a  sensi- 
tization.  The  amount  of  lecithin  required  for  complete  hsemoly- 
sis  is  determined  in  two  parallel  series,  one  on  the  addition  of 
small  amounts  of  cobra  venom,  the  other  with  large  amounts.  It  is 
found  that  far  more  lecithin  is  required  for  complete  solution  when 
there  is  a  large  excess  of  cobra  venom.  (See  Table  XI.) 


1  See  pages  209  et  seq.  3  See  pages  226  et  seq. 


310 


COLLECTED  STUDIES  IN   IMMUNITY. 


TABLE   XI. 


Amount  of 
Lecithin  Solu- 
tion Added. 

cc. 

1  cc.  5%  Ox  Blood  + 

0.4  cc.  5%  Cobra 
Venom. 

b 

0.1  cc.  0.1%  Cobra 
Venom. 

0.05 

0.035 
0.025 
0.015 
0.01 
0.0075 
0.005 
0.0035 

complete  solution 
moderate 
little 
faint  trace 
0 
0 
0 
0 

complete  solution 

it              it 

K              (i 
a              t( 
1  1              ii 

moderate 
trace 
0 

Now  if  the  cobra  venom  sensitized  the  blood-cells  for  the  lecithin, 
less  lecithin  would  be  required  for  solution  the  more  cobra  venom 
were  added.  As  a  matter  of  fact  the  reverse  is  the  case.  When  we 
used  a  large  excess  of  poison,  five  times  as  much  lecithin  was  re- 
quired for  complete  solution  as  when  smaller  doses  were  used.  This 
is  readily  explained  by  assuming  that  a  large  excess  of  amboceptor 
causes  a  deflection  of  the  lecithin,  a  phenomenon  which  we  have 
already  met  with  in  the  endocomplements. 

The  phenomena  observed  by  us  also  serve  to  explain  most  easily 
the  inhibiting  action  exerted  by  certain  sera.  As  is  well  known, 
lecithin  is  able  to  combine  with  albuminous  bodies,  sugars,  etc.  If 
this  union  is  so  firm  that  it  is  not  disrupted  by  the  affinity  of  the 
cobra  amboceptor,  it  will  be  impossible  for  the  lecithin  to  come  into 
action.  This  is  the  case,  for  example,  with  ox  serum,  which  when 
fresh  does  not  exert  a  trace  of  activation  on  goat  blood,  and  yet 
the  ox  serum  contains  sufficient  lecithin,  as  we  know  by  examining 
its  alcoholic  extract. 

Ox  serum  is  even  able  to  prevent  haemolysis  on  the  addition  of 
free  lecithin,  the  reason  being  evidently  because  it  contains  an  excess 
of  inhibiting  substances.  On  heating  the  serum  these  substances 
lose  their  action  to  a  greater  or  less  extent,  so  that  the  serum  is  able 
when  mixed  with  cobra  venom  to  effect  haemolysis.  As  already 
mentioned,  however,  the  hsemolytic  action  is  usually  considerably 
stronger  when  the  sera  are  heated  to  100°  C.  instead  of  only  to  65°  C. 
Perhaps  this  is  due  to  substances  possessing  different  degrees  of 
thermolability. 

In  other  cases  only  a  very  slight  difference  is  to  be  observed 


THE  MODE   OF  ACTION  OF  COBRA  VENOM.  311 

between  the  activating  power  of  fresh  and  of  heated  serum.  In 
this  case  evidently  the  fresh  serum  already  contains  free,  i.e.  active, 
lecithin,  and  the  inhibiting  substance  is  affected  but  to  a  slight 
degree  by  the  heating.  In  view  of  all  this  it  is  certainly  incorrect 
to  speak,  as  Calmette l  does,  of  a  definite  thermolabile  antibody 
which  is  destroyed  at  56°  C. 

It  is  natural  to  attempt  a  quantitative  estimation  of  the  cobra 
amboceptor  by  means  of  the  binding  of  lecithin;  also  to  think  of 
the  possibility  of  isolating  the  cobra  amboceptors  as  lecithids.  Ex- 
periments in  this  direction  are  now  under  way. 

The  results  of  the  experiments  here  given  furnish  a  further  in- 
sight into  the  nature  and  mode  of  action  of  the  amboceptors.  The 
demonstration  of  endocomplements,  as  well  as  the  significant  fact  that 
a  definite  chemical  and  crystalline  substance,  lecithin,  can  in  a  certain 
sense  play  the  role  of  complement,  would  appear  to  be  especially  im- 
portant for  the  development  of  our  knowledge  concerning  poisons. 

1  One  might  assume  that  the  haemolysis  by  cobra  venom  alone,  ascribed  in 
§  II  to  the  action  of  the  endocomplements,  was  caused  by  the  lecithin  contained 
in  the  red  blood-cells.  This  assumption,  however,  is  at  once  excluded  by  the 
fact  that  the  endocomplement  solutions  are  inactivated  by  heating  to  62°  C., 
showing  that  their  action  has  nothing  to  do  with  that  of  the  lecithin. 


XXVIII.  FURTHER  STUDIES   ON  THE  DYSENTERY 

BACILLUS.1 

By  Dr.  K.  SHIGA. 

WHEN  I  discovered  the  dysentery  bacillus  in  1897  I  found  that 
although  this  organism  apparently  remains  localized  in  the  intestine 
and  does  not  pass  into  the  circulation,  it  nevertheless  gives  rise  to 
the  development  of  specific  antibodies  in  the  serum.  This  fact, 
made  use  of  after  the  manner  of  the  Gruber-Widal  reaction,  furnished 
me  with  an  important  aid  in  the  diagnosis  of  the  dysentery  bacillus. 

In  the  course  of  the  following  years  the  facts  which  I  observed 
in  connection  with  epidemic  dysentery  have  been  confirmed  in  various 
parts  of  the  world,2  especially  since  Kruse  succeeded  so  well  in  his 
studies  on  this  disease  in  Germany.  To-day  there  is  no  longer  any 
doubt  concerning  the  identity  of  the  bacillus  isolated  by  Kruse 
with  mine,  even  though  there  is  still  a  slight  divergence  concerning 
certain  morphological  details.  All  of  the  important  character- 
istics of  the  bacilli  discovered  by  me,  as  well  as  their  agglutinat 
tion  by  serum  of  the  patients,  have  been  confirmed  by  Kruse.  Tha- 
certain  slight  differences  in  growth  may  occur  is  not  at  all  uncommon 
in  other  bacteria,  even  in  cholera.  The  question  as  to  the  presence 
of  motility  is  especially  hard  to  answer.  At  first  I  stated  that  my 
bacilli  were  motile;  Kruse 'found  them  immotile.  It  is  well  known 
that  it  is  not  always  easy  to  decide  whether  a  bacillus  is  motile  or 
not,  and  Kruse  himself  says  concerning  motility  as  a  characteristic 
of  the  coli  group  (Fliigge,  Vol.  II,  page  361)  that  "one  must  be  very 
careful  in  deciding  this  point,  for  the  movements  often  last  but  a  short 
time  and  are  not  present  under  all  conditions  of  life  (nutrient  medium, 

1  Reprint  from  the  Zeitsch.  f.  Hyg.  und  Infections-Krankheiten,  Vol.  41, 1902. 

2  Compare  also  the  study  published  since  this,  entitled  "Untersuchungen 
iiber die  Ruhr,"  Berlin,  1902. 

312 


FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.       313 

temperature,  etc.)."  In  this  connection  I  would  call  to  mind  the 
bacillus  of  erysipelas  of  swine,  whose  immotility  is  still  questioned 
by  many  observers.  I  have  always  described  the  motility  of  my 
cultures  as  feeble,  though  I  found  it  strange  that  I  was  unable  at 
first  to  demonstrate  flagella  by  staining  methods.  Later  on,  how- 
ever, I  succeeded  in  finding  two  terminal  flagella  in  one  preparation, 
and  thought  that  this  question  might  now  be  regarded  as  closed. 
To  what  extent  this  was  an  error  I  should  not  yet  like  to  say,  and 
for  the  present  I  should  also  not  like  to  regard  the  observations  of 
Vedder  and  Duval,1  who  found  peritrichal  flagella,  as  a  confirma- 
tion of  my  findings. 

In  1898  I  immunized  horses  with  dysentery  bacilli  and  obtained 
a  high-grade  serum  with  which  in  1898-1900  almost  three  hundred 
people  have  been  treated.  It  therefore  seemed  advisable  to  study 
this  dysentery  serum  from  the  standpoint  of  the  modern  theory 
of  immunity.  At  the  same  time  I  was  anxious  by  means  of  serum 
diagnosis  to  again  prove  the  identity  of  Kruse's  bacillus  with  mine. 

The  cultures  employed  were  the  following:  One  of  my  original 
cultures,  one  from  Prof.  Flexner,  one  culture  of  the  Kruse  bacillus 
from  the  Frankfurt  Institute,  and  a  Kruse  bacillus  from  Dr.  Conradi, 
Berlin.  I  may  at  once  say  that  in  all  the  various  bactericidal  experi- 
ments these  cultures  behaved  exactly  alike,  and  I  shall  therefore  in 
the  following  speak  of  the  dysentery  bacillus  as  such.  When  I  come 
to  speak  of  the  agglutination  I  shall  make  mention  of  certain  variations 
of  Flexner 's  bacillus  from  mine  and  Kruse's. 

To  begin,  the  bactericidal  action  of  normal  active  sera  was  tested 
on  the  dysentery  bacillus.  The  method  employed  corresponded 
exactly  to  that  described  by  M.  Neisser  and  Wechsberg,  to  whose 
paper  I  shall  therefore  refer.2 

The  amount  of  culture  planted  was  always  1/500  mg.  of  a  one- 
day  agar  culture,  and  in  the  dilution  employed  this  was  contained 
in  1.0  cc.  salt  solution.  The  total  amount  in  each  tube  was  always 
2.0  cc.,  to  which  quantity  three  drops  of  bouillon  were  then  added. 
The  serum  was  allowed  to  act  for  three  hours  at  37°  C.,  after  which 
time  six  drops  were  made  into  agar  plates.  In  judging  the  plates  we 
did  not  make  use  of  accurate  counting,  but  always  employed  the 

1  The  Etiology  of  Acute  Dysentery  in  the  United  States.    Journal  of  Experi- 
mental Medicine,  1902,  Vol.  VI.,  No.  2. 

2  See  pages  120  et  seq. 


314  COLLECTED  STUDIES  IN  IMMUNITY. 

method  of  Neisser  and  Wechsberg,  namely,  approximate  estimation, 
because  only  large  results  were  regarded  as  conclusive.  Frequently 
after  the  six  drops  had  been  taken  from  the  tube,  the  residue  was 
again  placed  into  the  incubator.  In  this  way  one  often  obtains 
valuable  confirmation  of  the  agar  plates  by  noting  whether  or  not 
there  is  a  growth  in  the  tubes. 

The  strongest  bactericidal  power  is  possessed  by  goat  and  sheep 
.sera,  but  this  is  but  slight  in  comparison  to  their  action  on  many 
other  species  of  bacteria.  0.3  cc.  of  these  sera  almost  completely 
killed  the  bacteria  under  the  conditions  mentioned.  Other  sera  are 
weaker,  such  as  ox,  horse,  human,  dog,  guinea-pig,  and  rabbit  serum. 
A  reactivation  of  normal  inactive  sera  succeeded  only  in  the  follow- 
ing combination:  normal  inactive  goat  serum  could  be  completely 
reactivated  by  normal  active  horse  serum  in  an  amount  which  by 
itself  did  not  kill  the  bacteria.  These  experiments  showed  that  only 
a  few  sera  could  be  used  for  reactivation  (e.g.  horse  serum)  apparently 
because  the  other  sera  did  not  contain  any  considerable  excess  of 
free  dominant  complement,  or  contained  none  at  all.  This  was 
entirely  confirmed  by  the  complementing  experiments  which  were 
made  with  a  high-grade  immune  serum.  The  immune  serum  used 
was  obtained  from  a  horse  which  I  myself  had  begun  to  immunize 
and  which  had  been  further  immunized  in  the  meantime.  The  serum 
was  sent  to  me  from  Japan  with  the  addition  of  0.5%  carbolic.  In 
the  small  amounts  in  which  the  serum  was  used,  this  addition  in  no 
way  disturbed  the  bactericidal  experiments,  as  was  shown  by  control 
tests.  The  first  experiments  undertaken  with  the  completion  by 
means  of  active  horse  serum  resulted  negatively  in  so  far  as  any 
destructive  action  was  concerned.  This  was  soon  found  to  be  due 
to  the  phenomenon  of  complement  deflection  described  by  Neisser 
and  Wechsberg;  for  when  smaller  and  still  smaller  doses  of  the  immune 
serum  were  employed  the  destructive  action  became  more  and  more 
marked.  Table  I,  in  which  column  A  gives  the  result  of  the  plate 
tests,  and  B  that  of  the  test-tube  experiment  made  at  the  same  time, 
shows  the  destructive  action  as  well  as  the  phenomenon  of  comple- 
ment deflection. 

From  this  it  is  seen  that  even  0.0025  and  0.0005  cc.  still  have  a 
distinct  bactericidal  action.  This  result  was  obtained  a  great  many 
times,  with  various  strains,  in  almost  the  same  manner. 

Besides  the  horse  serum  only  one  other  serum  could  be  used 


FURTHER  STUDIES   OX  THE   DYSENTERY   BACILLUS.       315 


for  complementing  the  immune  serum,  namely,  active  human  serum. 
Table  II  shows  an  experiment  with  this  serum. 


TABLE  I. 


Inactive 
Dysentery 
Serum,     cc. 

Active 
Horse  Serum, 
cc. 

Dysentery  Culture. 

A. 

No.  of  Colonies 
on  the  Plate. 

B. 

Growth  in  the 
Tubes. 

0.01 

0.3 

1.0cc.(  1/500  mg.) 

00 

+ 

0.0075 

0.3 

00 

+ 

0.005 

0.3 

00 

+ 

0.0025 

0.3 

almost  0 

0.001 

0.3 

0 

— 

0.00075 

0.3 

almost  0 

_ 

0.0005 

0.3 

about     50 



0.00025 

0.3 

«. 

100 

+ 

0.0001 

0.3 

n 

"     1000 

+ 

0.000075 

0.3 

it 

several  thous. 

+ 

0.00005 

0.3 

tf 

oo 

+ 

Control 


r—         0.3 
I  o.i 

I—  0.3 


1.0  cc.  (1/500  mg.) 


TABLE  II. 


several  thous. 

00 

0 
0 


Inactive  Dysen- 
tery Serum, 
cc 

Active  Human  Serum, 
cc. 

Dysentery  Culture. 

No.  of  Colonies  on  the 
Plate. 

0.01 

0.3 

1.0  cc.  (1/500  mg.) 

00 

0.003 

0.3 

00 

0.001 

0.3 

00 

0.0003 

0.3 

few 

0.0001 

0.3 

0 

0.00003 

0.3 

about     100 

0.00001 

0.3 

"      1000 

Control 


0.1 


0.3 


0.3 


1.0  cc.  (1/500  mg.) 


LTp  to  the  present  time  I  have  tested  the  serum  of  six  individuals 
and  found  it  active  in  five  cases  (four  times  in  placental  serum  and 
once  adult  serum) ;  only  once,  in  the  case  of  a  nephritis  patient,  was 
the  fresh  serum  ineffective  for  complementing.  It  may  be  mentioned 
that  one  of  these  sera  was  my  own,  and  this  was  considerably  stronger 
than  the  rest.  Whether  this  property  has  any  connection  with  an 
active  immunization  which  I  underwent  some  four  years  previously 
I  shall  leave  undecided. 


316 


COLLECTED  STUDIES  IN  IMMUNITY. 


I  believe  this  demonstrates  that  the  horse  immune  serum  em- 
ployed by  me  for  therapeutic  purposes,  meets  the  requirements  which 
are  nowadays  to  be  demanded  of  a  bactericidal  immune  serum, 
namely,  (1)  that  it  be  high  grade,  and  (2)  that  it  find  a  fitting  com- 
plement in  normal  human  serum.  This  is  the  first  serum  employed 
in  human  therapy  which  fulfils  the  conditions  laid  down  by  Ehrlich 
in  his  Croonian  Lecture,  1900.  The  excellent  curative  results  obtained 
by  me  in  Japan 1  furnish  abundant  confirmation  of  the  correctness  of 
Ehrlich 's  views. 

As  already  mentioned,  the  phenomenon  of  deflection  of  comple- 
ment could  be  demonstrated  very  prettily  with  the  complement  of 
this  active  horse  serum.  Since  this  deflection  is  primarily  dependent 
on  the  amount  of  immune  body  present,  it  may  perhaps  be  possible  to 
employ  the  degree  of  deflection  as  a  measure  of  the  titer  of  a  serum. 
Some  experiments  in  this  direction  which  I  have  undertaken  at  the 
suggestion  of  Prof.  M.  Neisser  have  not  yet  been  concluded. 

I  have  already  stated  that  the  other  active  sera  (e.g.  goat  serum, 
etc.)  could  not  be  used  for  complementing  the  dysentery  immune 
serum,  although  in  themselves  they  were  bactericidal.  But  for  this 
immune  serum  the  phenomenon  of  complement  deflection  can  be 
demonstrated  very  nicely  with  these  sera  also.  (See  Table  III.) 

TABLE  III. 


Dysentery  Immune 
Serum,     cc. 

Active  Goat  Serum, 
cc.   . 

Dysentery  Cultures. 

No.  of  Colonies  on  a 
Plate. 

0.1 
0.03 
0.01 
0.003 
0.001 

0.3 
0.3 
0.3 
0.3 
0.3 

1/500  mg. 

n 

(  t 
1  1 
t( 

00 
00 
00 

0 
0 

{0.3                       1/500  mg.                            0 
"                                                    00 
0.1                                                                                             0 
—                     0.3                               —                                 0 

Perhaps  also  this  method  of  testing  is  available  for  determining 
the  grade  of  bactericidal  sera. 

Furthermore  by  means  of  an  absorption  test  analogous  to  the 
experiments  of  A.  Lipstein  2  I  have  convinced  myself  that  the  deflec- 

1  Deutsche  med.  Wochenschrift,  1901,  Nos.  43-45. 
3  See  pages  132  et  seq. 


FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.       317 

tion  of  complement  described*  is  actually  due  to  an  excess  of  im- 
mune body  and  not,  for  example,  to  the  presence  of  an  anticomple- 
ment. 

Prof.  Neisser  and  I  thought  that  this  phenomenon  of  comple- 
ment deflection  could  be  utilized  in  another  direction.  Ehrlich  and 
his  pupils,  it  will  be  remembered,  have  demonstrated  the  existence 
of  a  plurality  of  complements.  In  view  of  this  it  was  conceivable 
that,  following  a  large  addition  of  inactive  immune  serum  to  a  normal 
serum  bactericidal  per  se,  only  that  complement  would  be  deflected 
which  is  able  to  complement  the  immune  serum,  while  the  remaining 
complements  were  left  unaffected.  From  this  it  would  follow  that 
the  normal  active  serum  in  question  would  in  the  main  have  lost 
only  this  one  bactericidal  action,  while  it  still  retained  almosj^all  the 
others.  One  would  thus  have  a  serum  which  had  lost  a  bactericidal 
action  chiefly  for  that  bacterium  whose  immune  body  has  been  added 
in  excess;  that  is  to  say,  a  truly  specific  nutrient  medium.  Proceeding 
from  these  considerations  we  first  infected  a  normal  stool  with  a 
small  quantity  of  dysentery  bacilli.  To  small  amounts  of  this  infected 
stool  2.0  cc.  normal  active  goat  serum  and  0.2  cc.  inactive  immune 
serum  were  added  and  the  mixture  kept  hi  the  thermostat.  At  the 
end  of  three  hours  six  drops  of  this  mixture  were  added  to  a  second 
tube  containing  2.0  cc.  normal  active  goat  serum  and  0.2  inactive 
immune  serum.  Agar  plates  were  made  (1)  from  the  original  infected 
stool;  (2)  from  the  first  tube;  (3)  and  from  the  second  tube  after  it 
also  had  been  kept  at  37°  C.  for  three  hours.  A  great  many  tests 
showed  that  a  specific  enriching  in  dysentery  bacilli  takes  place,  so 
that  when  the  first  plate  shows  only  a  few  scattered  colonies  of  dysen- 
tery bacilli,  Plates  II  and  III  show  numerous  colonies.  In  one  case 
we  evelw^ucceeded  in  finding  dysentery  bacilli  in  Plates  II  and  III, 
although  none  had  been  found  on  Plate  I.  It  may  be  mentioned 
that  we  used  the  agar  medium  recommended  by  v.  Drigalski  and 
Conradi l  for  the  diagnosis  of  typhoid  bacilli,  and  found  it  of  great 
advantage.  The  method  just  described  for  enriching  cultures  may 
perhaps  be  extended  and  perfected. 


1  Zeitschrift  fur  Hygiene,  Vol.  XXXIX. 


318  COLLECTED  STUDIES  IN  IMMUNITY. 

• 
Proagglutinoid. 

As  a  result  of  the  brilliant  investigations  of  Bail 1  on  the  one  hand 
and  of  Eisenberg  and  Volk2  on  the  other,  two  new  phenomena  have 
been  described  as  occurring  in  the  agglutination  reaction,  phenomena 
which  are  of  great  importance  in  the  study  of  agglutinins.  Bail 
first  showed  that  typhoid  bacilli  which  had  been  added  to  an  inac- 
tivated (by  heat)  agglutinin  and  then  centrifuged  could  not  longer 
be  agglutinated  by  the  addition  of  active  agglutinin.  The  study  of 
Eisenberg  and  Volk  described  an  irregularity  occurring  hi  a  series  of 
agglutinations  which  manifested  itself  in  this,  that  the  tubes  con- 
taining the  largest  amount  of  agglutinin  showed  only  feeble  agglutina- 
tion or  none  at  all,  while  the  tubes  containing  less  agglutinin  showed 
strong  agglutination.3  Bail  was  of  the  opinion  that  the  phenomenon 
observed  by  him  was  due  to  the  interaction  of  two  components  (cor- 
responding to  amboceptor  and  complement),  and  he  supported  this 
with  several  reactivating  experiments.  Eisenberg  and  Volk  explained 
the  irregular  course  of  the  agglutination  by  the  presence  of  agglutinoids, 
a  view  in  which  I  fully  agree. 

Following  Ehrlich's  nomenclature  I  should,  however,  like  to  term 
these  agglutinoids  4  proagglutinoids,  for  we  are  dealing  with  the  action 
of  substances  which  arise  from  the  agglutinins  as  a  result  of  external 
influences.  Furthermore  the  proagglutinoids  possess  a  higher  affinity 
for  the  bacilli  than  the  unchanged  agglutinin,  and  they  have  lost  that 
group  which  is  the  real  carrier  of  the  agglutinating  action,  while  the 
other  group,  which  effects  the  combination  with  the  bacteria,  is  left 
intact. 

1  Archiv.  f.  Hygiene,  1902,  Vol.  XLIII. 

2  Zeitschr.  f .  Hygiene,  1902,  Vol.  XL. 

3  This  paradoxical  phenomenon  is  mentioned  by  Asakawa  in  a  report  from 
the  Institute  for  Infectious  Diseases,  Tokio  (Sept.,   1901),  and  is  termed  by 
him  a  "reversely  behaving  phenomenon"  ("ein  umgekehrt  sich  verhaltendes 
Phanomen"). 

4  Since  the  conclusion  of  these  experiments  two  new  studies  have  appeared 
on  precipitoids.     R.  Kraus  (Centralblatt  fiir  Bakteriologie  1902,  Vol.  XXXII, 
No.  1),  v.  Pirquet  and  Eisenberg  (Extrait  d.  Bull.  d.  PAcade'rme  des  sciences 
de  Cracovie,   also  Centralblatt  f.   Bakteriologie,    1902,   Vol.  XXXI,   No.   15); 
also    Wiener    (Klin.    Wochenschr.    1901,    Uber    Precipitoide) .      The     authors 
arrive  at  the  same  results  as  have  been  described  for  agglutination.     Their 
experiments  for  demonstrating  these  precipitoids  are  similar  to  mine  for  the 
proagglutinoids. 


FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.       319 


From  the  large  number  of  experiments  which  I  have  made  with 
dysentery  and  typhoid  bacilli  I  have  selected  only  those  which  may 
serve  to  demonstrate  my  point.  Using  the  dysentery  immune  serum 
described  above  I  found  it  easy  to  demonstrate  the  Eisenberg-Volk 
phenomenon  both  with  my  original  dysentery  culture  and  with  a 
Kruse  culture.  The  method  was  as  follows:  An  agar  culture  wras 
suspended  in  10  cc.  of  an  0.85%  salt  solution.  At  first  this  was  used 
in  the  living  state;  later  on,  after  it  had  been  found  that  there  is  no 
difference  in  the  action  of  living  and  dead  culture,  the  culture  was 
used  with  the  addition  of  0.02  c.c  formalin  (40%).  One  cubic  centi- 
meter of  this  suspension  was  put  into  each  tube,  and  decreasing 
amounts  of  the  immune  serum  (2/io,  2/2o,  2/4o>  etc.,  usually  up  to 
2/5i2o)  added,  the  total  volume  in  each  of  the  tubes  being  2  cc.  The 
tubes  were  then  kept  in  the  thermostat  at  37°  C.  and  inspected  at 
the  end  of  2,  5,  and  24  hours,  both  with  the  naked  eye  and  with  a 
magnify  ing-glass.  The  results  were  noted  as  follows: 

no  agglutination; 
±         trace  agglutination; 
+         microscopically  distinct  but  feeble; 
+  +       very  distinct; 
+  +  +     entirely  clear  fluid  with  an  agglutinated  sediment. 

TABLE  IV. 


Dilution  of  the 
Dysentery 
Serum. 

Two  Hours. 

Five  Hours. 

Twenty-four 
Hours. 

1:10 

— 

— 

± 

1:20 

— 

± 

+  + 

1:40 

db 

+ 

+  +  + 

1:80 

+ 

+  + 

+  +  + 

1:160 

± 

+ 

+  +  + 

1:320 

— 

+ 

+  +  + 

1:640 

— 

± 

+  + 

1:1280 

— 

— 

± 

1:2560 

— 

— 

— 

1:5120 

— 

— 

— 

The  objection  was  made  that  the  agglutination  was  hindered 
in  the  low  dilutions  by  the  large  amount  of  serum  present  in  the 
tubes.  This  was  met  by  a  corresponding  addition  of  normal  serum, 
and  of  other  fluids  (gelatine,  mucilage,  etc.)  to  the  other  dilutions. 
In  the  old  dysentery  serum  the  question  as  to  the  development  of  the 


320 


COLLECTED  STUDIES  IN   IMMUNITY. 


proagglutinoid  from  the  agglutinin  could  only  be  answered  by  showing 
that  the  amount  of  proagglutinoid  already  present  in  this  serum  could 
be  increased  by  heating,  by  continued  exposure  to  light,  or  by  the 
addition  of  chloroform.  See  Table  V. 


TABLE  V. 


Dilution  of  the 

The  Serum  Exposed  to 
Light  for  17  Days. 

The  Serum  Heated  to 
60°  C.  for  One  Hour. 

The  Serum  Shaken  up 
with  Chloroform. 

Dysentery 

Serum. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

.:20 

- 

- 

- 

- 

- 

- 

- 

- 

- 

:40 

_ 

_ 

4- 

— 

— 

4- 

— 

— 

— 

:80 

± 

4- 

4-  4- 

— 

•  — 

4-  4. 

— 

— 

± 

:160 

-f. 

4- 

+  +  + 

— 

± 

4-4-4- 

— 

— 

4- 

1:320 

4- 

4- 

+  +  + 

— 

— 

4- 

— 

— 

± 

1:640 

'  ± 

± 

4-  -f 

— 

— 

± 

— 

— 

± 

1:1280 

— 

— 

— 

— 

— 

— 

— 

— 

— 

1:2560 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

— 

— 

1:5120 

— 

— 

— 

— 

— 

— 

— 

— 

— 

The  development  of  the  proagglutinoid  from  the  agglutinin  was 
still  more  distinct  in  a  fresh  typhoid  immune  serum  (goat).  This 
.serum,  which  had  shown  no  zone  of  proagglutinoid,  showed  a  dis- 
tinct zone  after  being  heated  twice  to  60°  for  four  hours. 

By  this  experiment  the  higher  affinity  of  the  proagglutinoid  is 
already  demonstrated.  It  can,  however,  be  confirmed  by  other 
experiments.  By  shaking  the  dysentery  serum  with  chloroform,  it 
was  possible  to  effect  almost  a  complete  transformation  of  agglutinin 
into  proagglutinoid  so  that  the  serum  hardly  agglutinated  in  any 
dilution.  When  to  a  dose  of  the  unchanged  dysentery  serum,  suffi- 
cient by  itself  to  effect  agglutination,  I  added  decreasing  amounts 
of  the  serum  treated  with  chloroform,  no  agglutination  was  obtained 
in  the  dilutions  up  to  1:160.  (Control  tests  with  chloroformed 
normal  serum  were  invariably  made.)  The  same  result  could  be 
obtained  with  dysentery  serum  that  had  been  heated.  Dysentery 
serum  heated  for  3  hours  to  65°  C.  was  able  in  dilutions  of  1 : 10  to 
1:320  to  prevent  agglutination  by  such  a  dose  of  the  unchanged 
dysentery  serum  which  by  itself  would  have  sufficed  to  agglutinate 
1:160.  (See  Table  VI.) 

Finally  it  remained  to  prove  that  the  proagglutinoid  had  really 
been  anchored  by  the  bacteria,  i.e.,  that  the  agglutinable  group  of 
the  bacilli  had  been  blocked.  This  was  readily  accomplished  by 


FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.       321 

centrifuging,  washing  the  bacilli  from  those  tubes  in  which  no  agglu- 
tination had  occurred,  and  adding  to  them  a  dose  of  agglutinin  which 
by  itself  would  suffice  for  agglutination.  The  result  was  that  these 
bacilli  always  showed  themselves  to  be  no  longer  agglu tillable, 
see  (Table  VII.) 

TABLE  VI. 


Dysentery 
Serum  Diluted, 
1:8. 

Dysentery  Serum 
Heated  to  65°  C.  for 
Three  Hours. 

Suspension 
of  Dysentery 
Cultures. 

2  Hours. 

5  Hours. 

0.1  CC. 

1:10      (1.0  cc.) 

1.00  cc. 

— 



1:20 

«  « 

— 

— 

1:40 

ft 





1:80 

(( 





1:160 

I  ( 





1:320 

(  I 

— 



1:640 

I  ( 

_ 

± 

1  :  1280 

(t 

_ 

+ 

1:2560 

l( 

_ 

+  + 

1:5120 

t  ( 

— 

+  + 

Control  0.1  cc. 

Salt  solution  1.0  cc. 

1.0  cc. 

+ 

+  + 

One  other  point  may  be  mentioned.  In  the  experiments  thus 
far  described  the  quantity  of  bacteria  was  the  same  in  all  the  tubes 
(See  above.)  However,  if  the  amount  was  greatly  increased,  other 
phenomena  were  observed.  Table  VIII  shows  that  the  zone  of 
proagglutinoid  disappears  entirely  if  a  sufficiently  large  quantity  of 
bacteria  are  employed. 

The  explanation  of  this  phenomenon  is  not  difficult  if  we  bear 
in  mind  the  experiments  of  M.  Neisser  and  Lubowsky  1  on  the  one 
hand  and  those  of  Eisenberg  and  Volk  on  the  other.  The  experiments 
of  the  latter  show  without  doubt  that  typhoid  bacilli,  for  example, 
are  able  to  anchor  a  far  greater  quantity  of  agglutinin  than  is  required 
for  their  agglutination.  One  may  therefore  assume  that  the  dysentery 
bacillus  also  possesses  a  large  number  of  receptors  which  are  able  to 
unite  with,  i.e.  anchor,  the  proagglutinoid.  The  occupation  of  only  a, 
few  of  these  many  receptors  by  the  active  agglutinin  is  apparently 
sufficient,  however,  to  agglutinate  the  dysentery  bacillus.  Hence  if 
we  add  comparatively  few  dysentery  bacilli  to  a  serum  which  contains 
much  proagglutinoid  and  little  agglu tinin,  a  large  number  of  receptors 
of  the  bacilli  will  be  occupied  by  proagglutinoid.  If,  on  the  contrary, 


1  See  pages  146  et  seq. 


322 


COLLECTED  STUDIES  IN   IMMUNITY. 


TABLE  VII. 
A. 


Dilution 

To  the  Residue 

of  the 
Dysentery 

24  Hrs. 

the    Dysentery 
Serum  (1/160) 

2  Hrs. 

5  Hrs. 

Remarks. 

Serum. 

is  Added. 

1:10 



T3 

Q} 

2.0  cc. 

— 

— 

1:20 

— 

Sp 

3 

«  « 

— 

— 

.. 

1:40 

+ 

IM 

•£ 

1:80 

+  +  + 

£H 

Not  tested  the  second 

1:160 

+  +  + 

8 

time  because  of  the 

1:320 

+  +  + 

a 

primary  agglutina- 

1:640 

+  + 

& 

tion 

1:1280 

+ 

3 

1:2560 

2 

+  + 

+  +  + 

J 

JS 

Control  2.0  cc. 

H 

+  dysentery 

bacilli 

+ 

+  +  + 

B. 


Dilution  of 
the  Serum 
Heated  to  65° 
for  3  Hours. 

5  Hours. 

24  Hours. 

The  Dysentery  Serum 
(1/160)  Added  to  the 
Residue. 

2  Hours. 

5  Hours. 

:10 

_ 

— 

1 

2.0oc. 

— 



:20 

— 

— 

j? 

— 

— 

:40 

— 

— 

— 

— 

:80 

— 

— 

4J 

c 

— 

— 

:160 

— 

— 

8 

— 

+  + 

:320 

— 

— 

H 

± 

+  +  + 

:640 

— 

— 

1 

+ 

+  +  + 

1:1280 

— 

— 

H 

+ 

+  +  + 

1:2560 

— 

— 

+  + 

+  +  + 

Control  2.0  cc. 

+  dysentery 
bacilli 

+ 

+  +  + 

TABLE  VIII. 


Dilution  of 

Normal  Suspension  of  Dysentery 
Bacilli. 

Five  Times  as  Strong  a  Suspen- 
sion of  Dysentery  Bacilli. 

the  Dysentery 

Serum. 

k 

2  Hours. 

5  Hours. 

24  Hours. 

2  Hours. 

5  Hours. 

24  Hours. 

1:10 

— 

_ 

± 

_ 

+  + 

+  +  + 

:20 

— 

± 

+ 

.       + 

+  + 

+  +  + 

:40 

± 

+ 

+  + 

+ 

+  + 

+  +  + 

:80 

± 

+ 

+  +  + 

+ 

+  + 

+  +  + 

:160 

± 

+ 

+  +  + 

± 

+ 

+  + 

:320 

± 

+ 

+  +  + 

— 

+ 

+  + 

:640 

— 

± 

+ 

— 

± 

+ 

:1280 

— 

— 

— 

— 

— 

— 

:2560 

— 

— 

— 

— 

— 

— 

1  :  5120 

~ 

_ 

~ 

~ 

FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.       323 


we  add  a  large  quantity  of  bacteria  to  the  same  amount  of  serum,  the 
proagglutinoid  will  not  suffice  to  occupy  all  the  receptors  and  some 
agglutinin  will  be  enabled  to  combine  with  the  bacteria.  This,  how- 
ever, results  in  agglutination. 

As  already  mentioned,  my  original  culture  proved  entirely  identical 
with  the  Kruse  culture  so  far  as  the  zone  of  proagglutinoid  was  con- 
cerned. The  Flexner  culture,  on  the  contrary,  behaved  differently, 
for,  although  it  was  agglutinated  in  the  same  degree  by  the  immune 
serum,  the  zone  of  proagglutinoid  was  entirely  absent.  This  is  well 
shown  in  the  following  table. 

TABLE  IX. 


Dilution  of  the 
Dysentery 
Serum. 

2  Hours. 

5  Hours. 

24  Hours. 

•20 

+  +  + 

+  +  + 

+  +  + 

:40 

-f  -j- 

+  +  + 

+  +  + 

:80 

-f 

+  +  + 

+  +  + 

:160 

± 

+  -f- 

+  + 

1:320 

_ 

+ 

-f 

1:640 

— 

4. 

+ 

1:1280 

— 

± 

+ 

1:2560 

— 

— 

— 

1:5120 

~ 

~~ 

— 

Absorption  tests,  which  were  then  made,  showed  that  the  Kruse 
bacillus  when  added  to  my  immune  serum  completely  abstracted  the 
agglutinin  and  proagglutinoid  for  this  strain,  while  the  agglutinin  for 
the  Flexner  strain  was  abstracted  to  only  a  slight  degree.  Conversely, 
when  the  Flexner  bacillus  was  added  to  my  immune  serum  and  the 
mixture  centrifuged  it  was  found  that  the  agglutinin  for  Flexner 's 
bacilli  had  been  completely  absorbed,  but  only  a  small  part  of  the 
agglutinin  and  proagglutinoid  for  the  Kruse  strain. 

We  shall  therefore  have  to  assume  that  my  original  strain  corre- 
sponds completely  to  the  Kruse  strain  so  far  as  the  receptor  apparatus 
is  concerned,  while  both  these  strains  possess  certain  receptors  identical 
with  those  of  Flexner's  strain,  and  others  which  differ  from  them. 
We  may  furthermore  assume  that  the  serum  with  which  these  experi- 
ments were  made  was  obtained  by  immunizing  not  only  with  my 
original  strain,  but  that  in  the  course  of  years  various  other  strains 
had  been  used  for  immunization.  In  this  way  agglutinins  of  various 
kinds  were  developed,  and  these,  of  course,  also  fitted  strains  wTith 


324  COLLECTED  STUDIES  IN  IMMUNITY. 

a  somewhat  different  receptor  apparatus.  It  may  be  remarked  that 
the  receptor  apparatus  of  the  bacteria  need  not  permanently  remain 
the  same  qualitatively  and  quantitatively,  as  is  well  shown  by  some 
experiments  of  mine  in  which  I  succeeded  in  producing  a  change  in 
these  properties  by  means  of  cultivation.  Thus  after  having  grown 
Kruse 's  bacilli  on  sterile  milk1  ten  consecutive  times  (always  trans- 
planting on  the  second  day),  and  finally  transplanted  it  to  agar, 
it  was  found  that  this  milk  strain  no  longer  showed  the  zone  of  the 
proagglutinoid  reaction. 

On  making  mutual  absorption  tests  it  was  seen  that  the  organism 
was  no  longer  like  the  original  Kruse  strain  but  entirely  like  that  of 
Flexner.  That  is  to  say,  this  cultivation  on  milk  had  effected  a 
gradual  change  in  the  Kruse  strain  which  manifested  itself  in  the 
changed  proagglutinoid  zone  of  the  absorption  power.  (See  Table  X.) 

It  remains  for  further  experiments  in  this  direction  to  see  whether 
I  shall  succeed  in  cultivating  the  Milk-Kruse  strain  back  to  the  original 
Kruse  strain,  or  in  changing  the  Flexner  strain  into  the  Kruse  strain. 
Thus  far  the  Flexner  strain,  as  well  as  the  Flexner  strain  altered 
by  cultivation,  have  preserved  their  properties  for  months. 

Resume. 

• 

1.  In  the  bactericidal  tests,  as  well  as  in  agglutination  reactions, 
my  original  dysentery  strain  from  Japan  proved  entirely  identical  with 
the  two  Kruse  cultures.     Since  these  are  the  most  refined  methods 
at  present  at  our  disposal,  there  can  be  no  doubt  as  to  the  identity 
of  my  original  cultures  of  1897  with  Kruse's  bacillus  of  1900. 

2.  The  dysentery  immune  serum  derived  from  a  horse  and  employed 
by  me  for  therapeutic  purposes  in  1898-1900  is  of  very  high  grade  and 

1  This  method  of  cultivation  was  really  made  because  of  the  statement  of 
Celli  ("Zur  Aetiologie  der  Dysenteric,  v.  Leydens  Festschrift")  that  my  bacillus 
would  also  coagulate  milk  like  the  bacillus  found  by  him,  if  it  was  transplanted 
8-10  times  on  alkaline  milk.  The  result  of  my  experiment  was  absolutely 
different,  for  neither  my  original  strain,  nor  the  strain  of  Kruse,  nor  that  of 
Flexner  coagulated  milk  when  the  cultures  were  grown  on  milk  ten  consecutive 
times,  provided  care  was  taken  to  protect  the  milk  from  contamination.  I  had 
already  tested  Celli' s  bacillus  in  Japan  and  found  that  it  produced  a  considerable 
amount  of  gas  and  coagulated  milk,  whereas  my  bacillus  does  not  do  this.  In 
view  of  this  and  of  the  further  fact  that  Celli' s  bacillus  does  not  agglutinate 
with  the  immune  serum  produced  by  means  of  my  bacillus,  I  conclude  that  these 
two  organisms  are  entirely  distinct  from  one  another — a  view  which  I  have 
already  expressed  in  a  previous  communication. 


FURTHER  STUDIES  ON  THE  DYSENTERY  BACILLUS.        325 

is  the  first  of  such  sera  whose  complementibility  by  human  serum  has 
been  proved. 

TABLE  X. 


Dilution  of 
the  Agglu- 

Normal Culture. 

First  Generation  of 
Milk  Culture. 

Fourth  Generation  of 
Milk  Culture. 

tinating 

Serum. 

2Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

1:10 





± 



± 

+ 

± 

+ 

+ 

1:20 

— 

± 

+  + 

± 

+ 

+ 

± 

+ 

+  -h 

1:40 

± 

+ 

+  +  + 

+ 

+  + 

+  +  + 

+ 

+  + 

+  +  -h 

1:80 

+ 

+  + 

+  +  + 

+ 

+  +  + 

+  +  + 

+ 

+  +  + 

+  +  + 

1:160 

± 

+ 

+  +  + 

± 

+  +  + 

+  +  + 

+ 

+  +  + 

+  +  + 

1:320 

— 

+ 

+  +  + 

± 

+  + 

+  +  + 

± 

+  + 

+  +  + 

1:640 

— 

± 

+  + 

— 

+ 

+  +  + 

± 

+ 

+  +  + 

1  :  1280 

— 

— 

± 

— 

± 

± 

— 

± 

+  + 

1:2560 

_ 

_ 

_ 

— 

— 

— 

— 

— 

— 

1:5120 

— 

— 

— 

~ 

~ 

Dilution  of 
the  Agglu- 

Sixth Generation  of 
Milk  Culture. 

Eighth  Generation  of 
Milk  Culture. 

Tenth  Generation  of 
Milk  Culture. 

tinating 

Serum. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

2  Hrs. 

5  Hrs. 

24  Hrs. 

1:10 

+ 

+  + 

+  + 

+  + 

+  +  + 

+  +  + 

+  + 

+  +  + 

+  +  + 

1:20 

+ 

+  + 

+  +  + 

+  + 

+  +  + 

+  +  + 

+  + 

+  +  + 

+  +  -h 

1:40 

+  + 

+  +  + 

+  +  + 

+  + 

+  +  + 

+  +  + 

+  + 

+  +  + 

+  +  -f- 

1:80 

+  + 

+  +  + 

+  +  + 

+ 

+  +  + 

+  +  + 

+  + 

+  +  + 

+  +  4- 

1:160 

+ 

+  +  + 

+  +  + 

+ 

+  + 

+  +  + 

+ 

+  +  + 

+  +  + 

1:320 

+ 

+  + 

+  +  + 

± 

+ 

+  +  + 

± 

+ 

+  + 

1:640 

± 

+ 

+  +  + 

— 

± 

+ 

— 

± 

+ 

1:1280 

_ 

•± 

+  + 

— 

— 

'  — 

_ 

_ 

_ 

1:2560 

_ 

— 

— 

— 

— 

— 

— 

— 

_ 

1:5120 

— 

— 

— 

— 

— 

— 

— 

— 

— 

3.  The  deflection  of  complement  of  Neisser-Wechsberg  could  very 
readily  be  demonstrated  with  this  serum  and  pointed  the  way  for  a 
new  method  of  specifically  enriching  bacterial  cultures  in  mixtures. 

4.  The  change  of  the  agglutinin  into  a  proagglutinoid  succeeded 
both  in  dysentery  serum  and  typhoid  serum. 

5.  Various   strains   may   possess   a   somewhat   different   receptor 
apparatus.     By  means  of  continued  culture  on  milk  a  certain  change 
in  the  behavior  of  the  receptor  apparatus  of  dysentery  bacilli  could 
be  effected. 


In  conclusion,  I  wish  to  express  my  thanks  to  Prof.  Ehrlich  and 
Prof.  M.  Neisser  for  aiding  me  in  this  study. 


XXIX.  METHODS   OF  STUDYING  ELEMOLYSINS. 

By  Dr.  J.  MORGENROTH,  Member  of  the  Institute. 

THE  object  of  the  following  article  is  to  give  a  brief  outline  of  the 
principles  governing  the  technique  of  hsemolytic  experiments.  It  may 
be  taken  for  granted  that  the  methods  employed  in  the  experiments 
already  described  will  be  applicable  to  many  problems  of  haemolysis 
still  to  be  studied  and  to  many  questions  concerning  bacteriolysins 
and  cytotoxins.  In  view  of  this  a  systematic  treatise  on  methods 
will  prove  of  considerable  value,  especially  to  one  who  uses  these 
methods  only  occasionally.  In  those  cases  where  a  particular 
technique  has  been  sufficiently  described  in  the  prevouis  papers. 
I  have  contented  myself  with  merely  giving  the  reference  to  this  paper, 

Aside,  however,  from  this  practical  object,  a  general  survey  of  the 
subject  is  to  be  given  which  will  show  how  a  system  of  technique, 
intelligently  built  up  on  a  comprehensive  theory,  has  made  it  possible 
to  push  our  analytical  inquiries  into  a  department  of  science  which 
formerly  constituted  a  sealed  book  to  the  ordinary  methods  of  chem- 
istry. Disregard  of  these  newer  methods  has  invariably  led  to  obscu- 
rity and  error,  as  we  have  been  able  to  show  on  several  occasions l  ; 
and  in  the  future,  even  if  refined  chemical  methods  can  successfully  be 
introduced  into  this  domain,  the  general  method  of  analysis  here 
outlined  will  always  form  the  basis  of  this  study.  According  to  our 
experience  the  study  of  haBmolysins  will  be  much  simplified  by  atten- 
tion to  a  number  of  technical  details  which  are  described  in  this 
article. 

I.  Collecting  and  Preserving  the  Blood  and  Blood  Serum. 

We  shall  begin  with  some  remarks  on  the  collection  and  preserva- 
tion of  the  blood  and  serum  required  for  these  experiments. 

As  a  general  rule  for  hsemolytic  experiments  it  is  not  necessary 

1  See,  for  example,  pages  181  et  seq.;  241  et  seq.;  283  et  seq.,  etc. 

326 


METHODS  OF  STUDYING  H^MOLYSINS.  327 

to  observe  aseptic  precautions;  usually  all  that  is  required  is  to 
collect  the  blood  in  dry  sterile  vessels,  avoiding  contamination  with 
dirt,  etc.     Hence  the  troublesome  method  of  collecting  blood  from 
the  carotid  of  the  animals  will  only  then  be  undertaken  if  for  some 
reason  asepsis  is  necessary  or   a   large   yield  of  blood   is  required. 
In  the  latter  case  the  yield  of  blood  can  be  considerably  increased 
toward  the  end  of  exsanguination  by  rythmic  compression  of  the 
cardiac  region.     With  goats,  sheep,  etc.,  the  blood  can  easily  be 
obtained  without  any  previous  dissection  by  means  of  a  suitable 
canula  thrust  through  the  skin  directly  into  the  jugular  vein  which 
has  been  distended  by  compression  on  the  cardiac  side.    This  is  the 
method  commonly  employed  in  obtaining  the  therapeutic  sera  from 
horses.     In  this  way  small  amounts  of  blood  can  be  drawn  from  the 
animals  a  great  many  times.     Smaller  animals,  such  as  dogs,  rabbits, 
guinea-pigs,  and  rats,  are  most  readily  bled  by  anaesthetizing  them,  dis- 
secting off  the  skin  of  the  thigh  and  then  with  one  stroke  cutting  both 
the  femoral  artery  and  vein.     From  rabbits  small  amounts  of  blood 
are  easily  obtained  by  incising  the  ear  with  a  scissors  or  by  means 
of  a  hypodermic  needle  introduced  into  the  marginal  ear  vein.     Small 
amounts  of  blood  can  be  obtained  from  birds  from  the  large  wing 
vein;  in  the  case  of  geese  and  ducks  the  web  of  the  foot  can  be  incised. 
For  purposes  of  obtaining  serum  the  blood  is  collected  in  cylindrical 
vessels  and  allowed  to  coagulate  spontaneously.     It  is  kept  hi  the 
refrigerator   until   the    serum   has   separated.     Several   hours   after 
collecting  the  blood,  it  is  well  to  loosen  the  clot  from  the  sides  of  the 
tube  by  means  of  a  glass  rod  or  spatula,  for  if  this  is  not  done  the 
serum  may  not  separate.     Small  amounts  of  blood  are  best  allowed 
to  clot  in  cylindrical  glasses  or  tubes  placed  slantingly.     After  clotting 
has  occurred  the  vessel  is  placed  upright.    The  serum  which  separates 
will  then  flow  to  the  bottom  and  can  be  poured  off  the  next  day. 
If  the  serum  is  clouded  with  blood-cells,  these  are  to  be  removed  as 
soon  as  possible.1 

When  the  serum  is  poured  off  the  first  time  the  vessel  containing 

1  An  excellent  centrifuge  with  a  capacity  up  to  200  cc.,  but  which  can  also 
be  had  for  larger  quantities,  is  that  made  by  Runne,  the  mechanic  in  Heidelberg 
University.  This  machine  is  made  either  for  water  or  electric  power,  and  runs 
exceedingly  smoothly.  For  centrifuging  smaller  quantities  of  fluid,  and  espe- 
cially for  sedimenting  blood-cells  from  dilute  blood  suspensions,  the  hand  cen- 
trifuge designed  by  Steenbeck-Litten,  and  made  by  F.  and  M.  Lautenschlager 
in  Berlin,  is  excellent. 


328  COLLECTED  STUDIES  IN  IMMUNITY. 

the  clot  can  be  kept  on  ice  for  24  hours  longer.     In  that  way  a  further 
yield  is  obtained. 

In  order  to  obtain  serum  immediately  the  blood  is  defibrinated 
by  whipping  it  with  a  stick  of  wood  or  by  shaking  it  in  a  bottle 
containing  some  glass  beads,  or  still  better  a  little  mass  of  dry 
sterilized  iron  turnings.  After  the  blood  is  defibrinated  it  is  centri- 
fuged  and  the  serum,  carefully  separated  by  means  of  a  pipette.  It 
is  well  to  fasten  a  long  rubber  tube  to  the  upper  end  of  the  pipette 
and  have  an  assistant  suck  while  one  watches  the  point  of  the  pipette. 

So  far  as  concerns  preservation  of  the  serum  it  may  be  said  that  our 
present  experiences  are  not  yet  sufficient  to  permit  us  to  formulate 
safe  rules  having  general  applicability.  It  is  not  only  necessary  to 
prevent  putrefaction,  but  also  to  preserve  intact  a  large  number  of 
most  unstable  substances,  the  conditions  necessary  for  whose  existence 
are,  in  part,  evidently  very  narrowly  limited.  Hence  for  the  present 
it  may  be  put  down  as  a  rule  that  in  all  important  primary  determina- 
tions only  very  fresh  serum  should  be  employed.  This  applies  above 
all  to  the  study  of  the  complements.  Negative  results  with  sera 
which  have  been  kept  several  days  and  which  have  been  exposed  to 
any  kind  of  thermic  or  chemic  influence,  are  particularly  unreliable. 
Hence  it  is  necessary  that  those  properties  of  a  serum  which  one 
purposes  to  study  should  be  examined  before  the  serum  is  preserved, 
so  that  secondary  changes  can  then  be  controlled  at  any  time. 

The  easiest  substances  to  preserve  are  the  antitoxins,  anticomple- 
ments,  antiamboceptors  and  the  majority  of  artificially-produced 
amboceptors.  By  the  addition  of  carbonic  acid,  Pfeiffer 1  has  succeeded 
in  keeping  a  cholera  immune  serum  derived  from  a  goat  for  five  years 
without  decrease  in  strength.  We  have  preserved  hsemolytic  ambo- 
ceptors for  a  long  time  without  any  addition,  by  keeping  the  sera  in 
an  ice-chest  at  8°  C.  The  development  of  bacteria  is  usually  prevented 
by  heating  the  serum  in  the  test-tubes  stoppered  with  cotton  plugs 
to  57°  for  half  an  hour.  In  this  way  the  serum  is  both  inactivated 
and  sterilized.  So  far  as  our  experience  goes  the  anticomplements 
and  antiamboceptors  can  be  preserved  in  the  refrigerator  like  the 
amboceptors.  Drying  the  serum  over  sulphuric  acid  or  over  anhy- 
drous phosphoric  acid  in  vacuum  can  also  be  used  for  these  substances. 

Of  all  the  substances  here  concerned  the  complements  are  by  far 
the  most  labile;  whenever  possible,  therefore,  fresh  serum  is  used 

1  See  Mertens,  Deutsche  med.  Wochensch.  1901,  No.  24. 


METHODS  OF  STUDYING  H.EMOLYSIXS.  329' 

for  activation.  Most  of  the  complements  will  keep  unchanged  for 
a  number  of  days  provided  the  serum  is  kept  on  ice.  But  this  does 
not  preclude  unpleasant  surprises,  diminutions  in  the  complementing 
power  often  occurring  to  a  high  degree  without  any  assignable  cause. 
According  to  our  experience  the  complements  of  guinea-pig  serum 
and  goat  serum  are  relatively  stable.  The  least  reliable  in  this  respect 
is  horse  serum,  whose  complementing  powers  are  often  partially  or 
completely  destroyed  within  twenty-four  hours.  The  complements 
also  suffer  when  the  serum  is  dried :  at  least  that  has  been  the  case  in  our 
rather  limited  experience. 

The  best  method  of  preserving  the  complements  for  a  long  time, 
and  the  one  almost  always  reliable  in  all  cases,  consists  in  freezing 
the  serum  at  — 10°  to  — 15°  C.  This  method  has  been  employed  in 
the  Institute  for  a  long  time.  The  serum  is  bottled  in  little  vials, 
which  are  then  kept  in  a  freezing  apparatus  or  in  a  well-insulated 
freezing  mixture  of  ice  and  salt,  each  vial  being  thawred  out  as  needed. 
This  procedure  is  at  present  the  only  one  which  is  of  general  appli- 
cability and  which  preserves  the  various  constituents  of  the  serum  for 
a  long  time. 

The  blood  used  for  the  hamolytic  tests  is  defibrinated  by  one  of 
the  methods  above  mentioned.  In  special  cases,  instead  of  defibrina- 
ting,  one  can  prevent  coagulation  by  precipitating  the  lime  salts. 
This  is  done  by  allowing  the  blood  to  flow  into  salt  solution  to  which 
citrate  of  soda  has  been  added,  as  was  recommended  by  Ehrlich.1 
For  the  majority  of  experiments  the  blood  is  diluted  with  physiological 
salt  solution.  If  for  any  reason  one  wishes  to  remove  the  serum,  the 
blood  is  separated  by  centrifuge  and  the  suspending  fluid  renewed 
several  times.  As  a  rule  blood  which  has  been  kept  on  ice  for  two 
days  can  still  be  used. 

It  should  also  be  mentioned  that  a  suitable  salt  solution  should 
be  employed  for  each  species  of  blood.  For  the  blood-cells  of  most 
mammals  a  feebly  hypertonic  solution  of  Nad  0.85%  is  best  adapted. 
In  0.85%  salt  solution  dog  and  horse  blood  frequently  shows  a  slight 
amount  of  spontaneous  ha3molysis  which  can  often  be  prevented  by 
using  a  somewhat  higher  concentration  (0.95%)  of  the  salt.  As 
a  rule  strongly  hypertonic  solutions  of  salt  are  to  be  avoided  because 
the  increased  contents  of  salt  markedly  inhibits  ha3molysis.2 

1  Ehrlich,  Fortschritte  der  Medizin,  1897,  Xo.  2. 
3  S,  Markl,  Zeitsch.  f .  Hygiene,  Vol.  39. 


330  COLLECTED  STUDIES  IN  IMMUNITY. 


II.  The  Method  of  Making  Haemolytic  Experiments. 
General  Considerations. 

With  a  little  practice  the  quantitative  estimation  of  haemolysis 
proves  very  simple.  The  two  fundamental  points,  entire  haemolysis 
(complete),  and  no  haemolysis  whatever  (0),  are  usually  very  readily 
recognized.  By  " trace"  we  mean  the  occurrence  of  a  faint  zone 
of  solution  observed  just  above  the  cells  by  gently  agitating  the 
test-tube.  The  estimation  of  complete  haemolysis  only  then  offers 
difficulties  if  considerable  agglutination  has  occurred,  so  that  the 
fluid  when  shaken  is  clouded  by  the  clumped  stromata.  Such  cases  in 
themselves  are  poorly  adapted  for  quantitative  studies  because  at 
times  the  rapid  agglutination  may  purely  mechanically  prevent  the 
escape  of  the  haemoglobin  and  so  simulate  an  absence  of  haemolysis. 

In  this  respect  according  to  our  experiences  the  greatest  diffi- 
culties are  presented  by  dog  blood-cells  and  the  specific  immune 
sera  (derived  from  goats)  against  these.  This  is  still  more  the  case  in 
such  sera  derived  from  rabbits.  It  often  happens,  before  even  a  trace 
of  haemolysis  has  occurred,  that  the  dog  blood-cells  are  agglutinated 
and  fall  to  the  bottom  of  the  test-tube.  Goose  blood  and  specific 
immune  serum  behave  similarly.  In  these  cases  it  is  necessary  by 
means  of  frequent  shaking  to  separate  the  agglutinated  blood-cells  so 
that  the  haemoglobin  is  given  chance  to  escape. 

In  those  cases  in  which  the  usual  method  of  describing  the  degree 
of  solution  does  not  suffice,  and  accurate  quantitative  determinations 
of  the  amount  of  blood-cells  dissolved  are  desired,  one  makes  use  of 
a  colorimetric  procedure  devised  by  Madsen  in  which  a  color  comparison 
is  always  made  by  dissolving  blood-cells  in  water.1 

Agglutination  is  usually  easily  recognized  on  shaking  up  the  sedi- 
mented  blood-cells.  It  becomes  very  evident  when  the  specimens 
of  blood  are  shaken  and  one  then  compares  the  rapidity  with  which 
the  blood-cells  settle  to  the  bottom.  This  is  always  greater  with 
agglutinated  blood-cells. 

In  general  a  5%  suspension  of  the  blood-cells  in  0.85%  salt  solu- 
tion has  proven  best  adapted  for  haemolytic  experiments.  1  to  2  cc. 
of  such  a  mixture  in  each  test-tube  is  sufficient  for  most  tests.  When 
material  is  scanty  one  can  use  amounts  very  much  smaller,  though 
usually  this  will  be  at  the  expense  of  accuracy.  In  this  case,  of 

1  See  Madsen,  Zeitschrift  fur  Hygiene,  Vol.  32,  1899. 


METHODS  OF  STUDYING  H^MOLYSINS.  331 

course,  the  test  is  made  in  very  narrow  test-tubes.1  The  serum 
to  be  tested  is  added  to  the  various  tubes  in  decreasing  amounts. 
The  volume  of  fluid  should  be  made  the  same  in  all  the  tubes  by 
the  addition  of  salt  solution,  for  the  total  amount  of  fluid  present 
may  influence  the  course  of  haBmolysis.  We  usually  keep  the  tubes 
in  a  thermostat  at  37°  C.  for  two  hours,  frequently  shaking  them  if 
necessary.  They  are  then  kept  in  the  refrigerator  at  8°  C.  overnight, 
which  allows  the  intact  blood-cells  to  settle.  In  the  cases  thus  far 
examined  by  us  this  method  has  always  sufficed  to  produce  the 
maximum  amount  of  haemolysis,  though,  of  course,  in  a  given  case 
it  may  have  to  be  modified  to  suit  the  circumstances. 

It  should  be  mentioned  that  in  testing  any  substances  for  haBmolytic 
action,  the  blood-cells  must  always  be  freed  from  serum  by  repeated 
washing,  for  the  serum  may  in  some  instances  (e.g.  with  solanin) 
give  rise  to  a  marked  inhibitory  action  and  so  lead  to  errors. 

III.  The  Technique  of  Immunization. 

So  far  as  the  production  of  hcemolytic  amboceptors  by  means  of 
immunization  is  concerned,  only  a  few  very  general  rules  can  be 
given,  for  thus  far  sufficient  systematic  investigations  have  not  been 
made  to  determine  the  optimal  conditions  in  any  one  direction.  In 
immunization  one  always  selects  such  animals  whose  serum  by  itself 
is  not  at  all  or  but  slightly  hsemolytic  for  the  blood  employed,  for 
then  the  development  of  a  hsemolysin  is  most  readily  determined 
and  the  normal  serum  of  this  species  always  furnishes  an  ideal  com- 
plement. If  animals  are  immunized  whose  serum  by  itself  already 
acts  hsemolytically  on  the  blood  used,  it  is  necessary  to  make  an 
exact  preliminary  determination  of  the  haemolytic  power  of  the 
normal  serum,  and  also  to  make  a  simultaneous  control  with  normal 
serum,  when  making  the  hsemolytic  experiments. 

In  some  instances  it  may  be  necessary  to  subject  the  blood  to  a 
preparatory  treatment,  for  the  purpose  of  removing  the  serum  more 
or  less  completely.  This  is  done  by  means  of  the  centrifuge  and 
is  required  especially  in  those  cases  in  which  intravenous  injections 
are  made,  or  if  large  amounts  of  a  blood  are  employed  whose  serum 

1  In  certain  cases  the  employment  of  very  high  columns  of  blood  is  indicated, 
for  in  that  case  the  development  of  zones  (colorless — feebly  red — strongly  red) 
permits  of  a  very  accurate  estimation  of  the  period  of  incubation  of  the  poison, 
or  of  the  different  vulnerability  of  the  blood-cells.  See  also  Madsen,  I.e. 


332  COLLECTED  STUDIES  IN  IMMUNITY. 

is  highly  toxic  for  the  animal  injected.  If,  for  example,  a  rabbit  is 
injected  intravenously  with  10  cc.  of  dog  blood  whose  serum  has 
not  previously  been  removed,  the  animal  will  die  acutely.  By  pre- 
viously heating  the  serum  one  also  obviates  the  reactive  production 
of  serum  coagulins  and  anticomplements,  both  of  which  can  at  times 
hinder  the  estimation  of  haemolysis.  A  general  rule  as  to  which 
mode  of  injection  is  to  be  chosen  for  immunization  cannot  be  laid 
down.  Larger  laboratory  animals  are  usually  injected  subcutaneously  ; 
goats  usually  bear  intraperitoneal  injections  very  well.  This  mode 
of  injection,  using  blood-cells  which  have  previously  been  dissolved 
with  water,  is  used  especially  when  a  particularly  marked  "  ictus 
jmmunisatorius  "  is  desired,  as,  for  example,  in  the  production  of 
isolysins.  Birds  are  injected  into  the  large  pectoral  muscles  or 
intraperitoneally.  For  rabbits  and  guinea-pigs  the  intraperitoneal 
injections  are  well  adapted,  since,  if  the  material  is  not  positively 
sterile,  secondary  injections  (which  in  subcutaneous  inoculations 
often  lead  to  troublesome  abscesses,  especially  in  the  rabbit)  are 
most  readily  avoided.  Injuries  to  the  intestine  are  best  avoided  by 
holding  the  animals  almost  vertically,  head  down,  and  thrusting 
the  needle  into  the  abdomen  in  the  median  line  a  little  above  the 
bladder.  The  needle  should  not  be  too  sharp,  nor  thrust  in  very 
deeply.  (Personal  communication  of  Dr.  R.  Krause.)  The  repetition 
of  intravenous  injections  offer  especial  difficulties,  for  after  hsemolysin 
formation  has  once  occurred  the  blood-cells  introduced  are  rapidly 
dissolved,  leading  to  the  death  of  the  animal  from  embolism. 
(Rehns.1) 

Another  thing  which  may  lead  to  death  from  embolism  is  the 
formation  of  coagulins  in  consequence  of  a  previous  injection  of 
blood  which  has  not  been  freed  from  serum.  These  coagulins  cause 
a  rapid  formation  of  precipitates  within  the  blood  circulation.2 

The  amount  of  blood  used  depends  upon  the  size  of  the  animal 
to  be  injected  and  upon  the  special  conditions  of  the  experiment. 
Up  to  a  liter  of  blood,  freed  from  most  of  its  serum,  can  be  injected 

1  Rehns,  Comp.  rend,  de  la  Soc.  de  Biol.  1901,  No.  12;  see  also  similar 
observations  made  on  man  by  Bier,  Munch,  med.  Wochensch.  1901,  No.  15. 

'Very  likely  the  inexplicable  results  obtained  by  Magendie  ("Vor- 
lesungen  iiber  das  Blut,"  German  translation  by  Kriipp,  Leipzig,  1839)  were  due 
to  the  formation  of  coagulins.  Magendie  found  that  rabbits  which  had  tolerated 
two  intravenous  injections  of  egg  albumin  without  any  injury  whatever  immedi- 
ately succumbed  to  a  further  injection  made  after  a  number  of  days. 


METHODS  OF  STUDYING  HJEMOLYSINS. 

into  goats  without  injury.  In  rabbits  of  2  kilos  it  will  hardly  be 
possible  to  go  beyond  100  cc.;  and  guinea-pigs,  corresponding  to 
their  weight,  proportionately  less.  According  to  our  experience  a 
single  injection  of  20-30  cc.  sheep,  goat,  ox,  or  dog  blood  leads  to  a 
strong  formation  of  hsemolysin,  which  can  be  still  further  increased 
by  a  subsequent  injection  of  40-60  cc.  six  to  ten  days  later.  We 
have  found  that  further  injections  of  the  same  or  larger  amounts 
(80-100  cc.)  have  no  advantage.  We  have  occasionally  observed 
that  these  were  associated  with  a  decrease  in  the  amount  of  hsmolysin. 
As  a  rule,  the  serum  attains  its  maximum  power  between  the  sixth 
and  tenth  day,1  but  this  is  subject  to  individual  variations,  as  is  shown 
by  the  case  described  by  Ehrlich  and  Morgenroth  of  a  goat  in  which 
an  isolysin  developed  critically  on  the  fifteenth  day  (see  page  29). 

The  injections  of  serum  lead  principally  to  the  production  of 
antiamboceptors  and  anticomplements,  in  some  instances  also  to  that 
of  haemolytic  amboceptors  in  consequence  of  the  receptors  present 
in  solution  in  the  serum.2  The  production  of  antiamboceptors 
necessitates  a  special  selection  of  the  animal  species.  Our  own 
positive  results  are  limited  to  the  injection  of  goats  either  with  the 
serum  of  a  rabbit  which  had  been  immunized  with  ox  blood,  or  with 
an  isolytic  serum.  Since  in  these  cases  the  immune  serum  is  toxic 
for  the  goat,  or,  more  particularly,  acts  destructively  on  the  blood, 
it  is  necessary  to  commence  with  the  injection  of  small  amounts 
(10-20  cc.)  and  gradually,  as  the  reaction  subsides,  go  on  to  larger 
doses.  As  in  the  case  of  all  immunizations  with  toxic  substances  it  is 
particularly  necessary  to  keep  a  careful  control  of  the  weight  of  the 
animals;  the  rule  always  to  be  observed  is  that  immunization  can 
only  then  be  proceeded  with  when  the  animal  has  again  attained  the 
weight  it  originally  possessed. 

In  order  to  produce  anticomplements  larger  animals,  such  as  sheep 
and  goats,  are  injected  with  increasing  amounts  of  normal  serum, 
beginning  usually  wTith  fairly  large  amounts — 100  to  500  cc.  As  a 
rule,  when  rabbits  have  been  injected  two  or  three  times  with  guinea- 
pig,  horse,  goat  or  ox  serum  (commencing  with  5  to  10  cc.  and  increas- 
ing to  20  to  50  cc.),  a  plentiful  supply  of  anticomplement  will  have 
developed  in  the  serum.  In  many  cases  the  injection  of  an  inactive 


1  See  also  Bulloch,  Centralblatt  f.  Bact.,  Vol.. 29,  1901. 

2  See  Morgenroth  (page  241,  this  volume)   and  P.  Miiller,  Munch,  med. 
Wochensch.  1902,  Xo.  32. 


334  COLLECTED  STUDIES  IN   IMMUNITY. 

serum,  which  had  thus  been  deprived  of  much  of  its  toxic  property, 
would  appear  to  be  preferable,  for,  owing  to  the  complementoids 
which  it  contains,  this  would  cause  the  production  of  anticomplements 
just  as  well  as  fresh  serum.  (See  pages  79  et  seq.) 

If  it  is  desired  by  the  injection  of  a  certain  serum  to  produce 
anticomplements  which  are  also  directed  against  various  other  sera,1 
it  is  necessary  to  repeat  the  injections  several  times  in  increasing 
amounts.  While  treating  a  goat  with  rabbit  serum,  Ehrlich  and 
Morgenroth  observed  the  development  first  of  anticomplements 
directed  exclusively  against  the  complement  of  rabbit'  serum  (iso- 
genic  anticomplements);  in  course  of  time  anticomplements  directed 
against  the  complements  of  guinea-pig  serum  (alloiogenic  anticom- 
plements) also  appeared.  Here  evidently  we  are  dealing  with  partial 
complements,  present  in  rabbit  serum  in  small  amounts,  which  require 
several  repetitions  of  the  injections  in  increasing  amounts  in  order 
to  excite  the  production  of  anticomplements. 

In  the  production  of  serum  coagulins  [precipitins]  one  proceeds  as 
for  anticomplements.  These  serum  coagulins  have  been  shown  to 
possess  considerable  value  for  the  forensic  determination  of  various 
species  of  blood,  especially  human  blood,  as  has  been  shown  by  the 
researches  of  Wassermann  and  Schiitze,  Uhlenhuth,  and  many  others. 
In  the  preduction  of  milk  coagulins  one  or  two  injections  of  20  to 
40  cc.  of  milk  into  a  rabbit  are  usually  sufficient.  The  milk  can  be 
heated  to  60°  previous  to  injection  in  order  to  reduce  the  number  of 
germs  present.  In  connection  with  the  production  of  serum  coagu- 
lins Uhlenhuth  makes  some  interesting  statements  (Deutsch.  med. 
Wochenschr.  1902,  No.  37).  Among  other  things  he  describes 
something  we  had  also  noticed,  namely,  the  occasional  failure  of  the 
reaction  and  the  development  of  "alloiogenic  "  coagulins  as  the 
titer  of  the  serum  increased,  a  fact  which  corresponds  to  what  we 
have  above  described  for  the  formation  anticomplements.2 

IV.   Determining  the  Haemolytic  Action. 

The  fact  that  certain  poisons  of  vegetable  or  animal  origin,  as 
well  as  normal  sera  and  other  body  fluids,  possess  a  haemolytic  action 
can  be  determined  so  readily  that  it  will  be  superfluous  to  enter  further 

1  See  pages  111  et  seq. 

2  Concerning    isogenic    and    alloiogenic    anticomplements,    see    Morgenroth 
and  Sachs,  pages  258  et  seq. 


METHODS  OF  STUDYING  H.EMOLYSIXS.  335 

into  the  subject.  In  passing,  however,  it  may  be  mentioned  that 
for  an  investigation  in  this  direction  to  be  at  all  complete  it  is  necessary 
to  make  use  of  as  many  different  species  of  blood-cells  as  possible. 
The  susceptibility  of  the  cells  can  be  extraordinarily  diverse,  so  that 
certain  poisons  exert  a  marked  hamolytic  effect  on  some  species  of 
blood,  while  they  fail  to  have  any  action  whatever  on  other  species. 
Thus  the  poison  of  the  garden  spider,  studied  by  Sachs,1  is  inert  for 
guinea-pig  or  dog  blood-cells,  while  it  has  strong  haemolytic  powers  for 
rabbit  blood-cells.  Crotin  which  dissolves  certain  blood-cells  (e.g. 
rabbit  blood)  and  agglutinates  others  (e.g.  hog  blood)  behaves  in 
similar  fashion.2 

In  the  case  of  the  specific  hsemolysins  produced  by  immunization 
the  choice  of  blood,  of  course,  is  already  indicated.  But  even  here, 
extending  the  investigations  to  numerous  other  species  of  blood 
may  lead  to  valuable  information  concerning  a  community  of  recep- 
tors such  as  exists  between  sheep,  goat,  and  ox  3  and  as  has  recently 
been  showrn  by  Marshall  to  exist  between  man  and  certain  species  of 
monkeys.  In  testing  a  serum  for  the  presences  of  isolysins  it  is 
necessary  to  use  the  blood  of  numerous  individuals,  for  according  to 
our  experience  the  sensitiveness  of  the  blood,  in  the  case  of  goats, 
is  subject  to  the  widest  individual  fluctuations.  In  this  way  one 
can  easily  be  misled  to  assume  that  the  experiment  results  nega- 
tively. It  is  advisable,  when  testing  a  fluid  for  haemolytic  properties 
for  the  first  time,  to  remove  the  serum  by  washing  the  blood-cells  at 
least  once.  Under  certain  circumstances  a  slight  degree  of  hsemolytic 
action  can  be  masked  by  an  antihaemolytic  action  of  the  normal  serum. 
This  is  seen  to  a  high  degree  in  the  case  of  the  haemolytic  poisons 
of  the  organ  extracts.4  So  far  as  the  dosage  is  concerned  one 
should  select  wide  limits,  especially  in  the  first  experiments.  If 
one  has  once  determined  the  presence  of  a  ha?molytic  action,  the 
quantitative  estimation  follows  by  means  of  a  more  or  less  finely 
graded  series  of  experiments.  Types  of  these  experiments  are  found 
on  pages  168,  270,  276,  etc. 

In  testing  a  haemolysin  which  has  not  yet  been  examined,  it  is 

1  See  pages  167  et  seq. 

2  Elf  strand,  Uber   giftige    Eiweissstoffe  welche    Blutkorperchen    verkleben. 
Upsala,  1891. 

3  See  pages  93  et  seq. 

4  See  Korschun  and  Morgenroth,  pp.  267  et  seq. 


336  COLLECTED  STUDIES  IN  IMMUNITY. 

important  to  determine  whether  the  hsemolytic  agent  is  a  haptin 
in  the  true  sense.  So  far  as  the  alkaloids,  glucosides,  etc.,  which 
act  hsemolytically  are  concerned,  they  are  generally  readily  identi- 
fied by  means  of  the  chemical  methods  devised  for  their  separation, 
methods  based  on  precipitations  and  shaking  out  with  solvents. 
This  is  not  true  for  the  haptins;  they  cannot  be  prepared  by  these 
methods.  At  the  most,  it  is  possible  to  precipitate  them  in  con- 
junction with  the  albuminous  bodies.  Another  distinction  consists 
in  this,  that  the  substances  which  are  chemically  defined  are  usu- 
ally thermostable,  while  the  haptins  in  the  great  majority  of  cases 
are  destroyed  by  heat,  especially  by  boiling  temperature.  One 
distinction  above  all,  however,  is  the  fact  that  only  the  haptins  are 
capable  of  causing  the  production  of  antibodies  by  immunization, 
.and  this  makes  a  classification  possible  even  in  difficult  cases.  Fre- 
quently the  facts  which  we  have  already  learned  about  a  substance 
allow  us  to  make  definite  conjectures.  For  example,  if  a  vegetable 
extract  possesses  hsemolytic  properties  which  are  not  destroyed  by 
boiling,  and  if  it  is  found  that  the  hsemolytic  substance  is  soluble 
in  ether,  we  can  at  once  exclude  this  from  the  class  of  haptins.  On 
the  other  hand,  if  one  finds  that  the  hsemolytic  action  of  an  animal 
body  fluid  is  destroyed  by  heating  to  56°  C.,  this  fact  already  argues 
in  favor  of  a  haptin;  Other  methods,  including  perhaps  the  immu- 
nizing reaction,  would  then  be  required  to  determine  this  positively. 


V.   The  Study  of  Complex  Haemolysins. 

We  now  take  up  a  question  of  paramount  importance  which 
arises  in  the  study  of  every  hsemolytic  poison,  namely,  whether  in 
any  given  instance  we  are  dealing  with  a  simple  hsemolysin,  or  with  a 
complex  one  consisting  of  amboceptor  and  complement. 

In  determining  the  complex  nature  of  a  hsemolysin  we  now  have 
the  following  methods  at  our  disposal: 

1.  Separation  of  amboceptor  and   complement  by  allowing  the 
former  to  be  tied  by  red  blood-cells  at  low  temperatures. 

2.  Removal  of  the  complement  or  changing  the  same  into  the 
inert  complementoid. 

(a)  Absorbing  the  complement  by  means  of  certain  cells  (e.g., 
yeast-cells,  bacterial  cells,  cells  of  animal  organs),  or  by  means  of 
porous  filters. 


METHODS  OF  STUDYING  ILEMOLYSINS.  337 

(b)  Thermic  and  chemic  influences,  such  as  heating  to  50-60°  C., 
the  action  of  alkalies  and  acids,  digestion  with  papayotin. 

The  separation  of  amboceptor  and  complement  at  low  tempera- 
tures is  of  the  utmost  importance  and  has  been  used  for  the  analysis 
of  complex  hsemolysins  with  considerable  success.  The  conditions 
necessary  for  the  successful  operation  of  this  method  have  been 
discussed  in  detail  in  a  previous  paper.  A  separation  is  only  then 
possible  when  at  low  temperatures  the  affinity  between  the  cyto- 
phile  group  of  the  amboceptor  and  the  receptor  is  greater  than  that 
between  the  complementophile  group  of  the  amboceptor  and  the 
corresponding  group  of  the  complement.  The  degree  of  difference 
in  the  affinities  would,  of  course,  determine  the  degree  of  complete- 
ness of  the  separation.  In  some  instances  most  peculiar  relations 
are  found,  as  is  shown,  for  example,  by  the  behavior  of  eel  serum 
to  rabbit  blood.  Attempts  to  effect  separation  at  low  temperatures 
fail  in  this  case,  first,  because  haemolysis  ensues  even  at  0°  C.,  and 
second,  because  the  employment  of  higher  concentrations  of  salt 
(up  to  5%),  which  in  other  cases  has  afforded  a  means  of  loosening 
the  combination  of  amboceptor  and  complement,  does  not  suffice 
to  prevent  haemolysis.  Naturally  from  this  behavior  we  must  not 
conclude  that  eel  serum  does  not  contain  a  complex  hsemolysin,  but 
merely  that  in  this  case  peculiar  conditions  are  present  which,  owing 
to  the  insufficiency  of  the  methods  thus  far  employed,  are  still  obscure 
to  us.  In  those  cases  in  which  separation  at  low  temperatures  fails, 
a  second  method  may  be  considered.  This  depends  on  the  fact 
that  a  high  degree  of  salt  concentration,  somewhat  after  the  manner 
of  low  temperatures,  can  prevent  haemolysis;  concentrations  which 
still  permit  the  union  of  receptor  and  amboceptor  preventing  that 
of  amboceptor  and  complement.  The  prevention  of  haemolysis  by 
means  of  salts,  first  described  by  Markl 1  and  erroneously  ascribed 
by  him  to  conditions  of  diffusion,  is  also  due  to  this.  Markl  entirely 
overlooked  the  fact  that  in  certain  cases  the  combination  of  toxin 
and  antitoxin  (e.g.  Tetanus  toxin + Antitoxin)  is  also  prevented  by 
salt.  (Knorr.)  For  the  application  of  this  method  see  Ehrlich  and 
Sachs,  page  214. 

It  is  perfectly  obvious  that  the  cold  method  will  fail  absolutely 
in  cases  like  the  one  described  by  Ehrlich  and  Sachs  (page  217)  in 
which  the  union  of  amboceptor  and  complement  is  the  prerequisite 

1  Markl,  Zeitschr   fur  Hygiene,  Vol.  39,  1902. 


338  COLLECTED  STUDIES  IN  IMMUNITY. 

for  the. union  with  the  blood-cells.  Such  a  possibility  must  always 
be  borne  in  mind. 

The  technique  of  this  separation  at  low  temperatures  is  very 
simple.  The  tubes  containing  the  blood  and  the  serum  respectively 
are  cooled  to  0°  by  being  placed  in  iced  water  or  by  packing  in  ice. 
Thereupon  the  serum,  in  amounts  which  are  not  far  either  way  from 
the  single  solvent  dose,  is  added  to  the  blood.  After  being  kept 
at  0°  for  two  hours  the  mixture  is  rapidly  centrifuged  and  the  super- 
natant fluid  quickly  removed.  If  desired,  the  sedimented  blood- 
cells  can  be  washed  with  salt  solution  and  then  suitably  suspended. 
The  decanted  fluid  is  again  mixed  with  blood-cells.  For  this  pur- 
pose, in  order  not  to  increase  the  total  volume,  one  takes  the  blood- 
cell  sediment  centrifuged  from  the  required  amount  of  the  5%  sus- 
pension. If  a  complete  separation  of  amboceptor  and  complement 
has  been  effected,  it  will  be  found  that  neither  are  the  sedimented 
blood-cells  dissolved  nor  is  the  decanted  fluid  able  to  dissolve  the 
blood-cells  added  anew.  It  is  then  necessary  to  determine  the  pres- 
ence of  complement  in  the  decanted  fluid,  which  is  done  by  adding 
suitable  amounts  of  serum  inactivated  by  heating.  Similarly  the 
amboceptor  anchored  by  the  blood-cells  at  low  temperatures  is  demon- 
strated by  adding  to  the  sediment  the  complement  present  in  the 
decanted  fluid. 

The  second  and  simpler  method  is  that  of  inactivating  the  hsemo- 
lytic  serum  by  means  of  heat  and  then  activating  the  amboceptor 
by  the  addition  of  complement.  In  this  the  chief  difficulty  often 
consists  in  the  fact  that  a  certain  complement  required  in  a  par- 
ticular instance  is  not  contained  in  all  sera,  and  further  that  the  sera 
which  contain  this  particular  complement  often  in  themselves  dis- 
solve the  blood-cells  by  means  of  a  normal  amboceptor. 

There  are  several  ways  of  overcoming  these  difficulties.  The 
neatest  method  and  one  which  is  applicable  in  many  cases  consists 
in  selecting  as  the  complementing  agent  the  serum  of  that  animal 
species  whose  blood  is  being  tested,  as,  for  example,  using  guinea-pig 
serum  as  complement  for  amboceptors  acting  on  guinea-pig  blood. 
In  such  cases  a  solution  of  the  blood-cells  by  means  of  the  animal's 
own  serum  is,  of  course,  precluded. 

In  all  other  cases  one  must  make  use  of  complementing  sera 
which  are  unrelated  to  the  species  of  blood  in  question.  One  fre- 
quently discovers  sera  for  this  purpose  which  do  not  in  themselves 
dissolve  the  blood-cells  to  be  tested,  as,  for  example,  in  reactivating 


METHODS  OF  STUDYING  HLEMOLYSIXS.  339 

the   amboceptors    for  sheep   blood  or  ox  blood   by  means  of  goat 
serum. 

But  it  is  often  possible  to  complement  an  amboceptor  with  a 
serum  which  in  itself  dissolves  the  blood-cells,  but  which,  in  the 
amounts  in  which  it  is  able  to  effect  completion,  has  little  or  no- 
haemolytic  action.  It  is  obvious  that  in  such  cases  the  solvent  power 
of  the  serum  by  itself  must  be  accurately  determined  by  means  of 
controls.  While  this  method  is  often  successful,  the  relation  in  these 
sera  between  the  normal  amboceptor  and  the  complement  is  fre- 
quently so  unfavorable  that  it  is  impossible  to  complement  the 
foreign  amboceptor.  In  such  cases  one  can  get  rid  of  the  normal 
amboceptor  by  anchoring  it  to  blood-cells  at  low  temperatures,  as 
Flexner  and  Noguchi l  have  recently  done  in  order  to  obtain  com- 
plements for  the  haemolytic  amboceptors  of  snake  venoms.  Or  one 
can  attempt  artificially  to  increase  the  amount  of  complement  con- 
tained in  complementing  serum,  after  the  method  of  P.  Miiller.2 
This  author  succeeded  in  effecting  a  considerable  increase  in  the 
complements  of  chicken  serum,  by  injecting  the  animals  with  solu- 
tions of  peptone. 

So  far  as  the  choice  of  the  complementing  sera  is  concerned  it  is 
obvious  that,  in  amboceptors  produced  by  immunization,  whenever 
possible  the  preference  will  be  given  to  those  sera  which  are  derived 
from  the  same  species  which  yielded  the  amboceptor.  For  the 
remaining  cases  the  principle  may  be  formulated  that  that  serum 
is  most  useful  which  is  derived  from  a  species  closely  related  to  that 
furnishing  the  amboceptor,  because  often  in  distantly  related  species 
partial  amboceptors  present  only  in  very  slight  amounts  are  com- 
plemented.3 

Another  point  of  considerable  importance  in  the  completion  of 
amboceptors  is  the  manner  in  which  the  sera  are  inactivated.  As 
a  rule  inactivation  is  effected  by  heating  the  serum  for  half  an  hour 
in  a  water-bath.  According  to  recent  investigations  special  atten- 
tion must  be  paid  to  the  degree  and  duration  of  this  action.4 

1  Flexner  and  Xoguchi,  Journal  of  Exp.  Medicine,  Vol.  VI,  1902. 
:  Miiller,  Centralblatt  f.  Bacteriologie,  Orig.  Vol.  29,  1901. 

3  Ehrlich  and  Morgenroth,  see  pages  110  et  seq. 

4  In  order  accurately  to  observe  the  temperature  constantly  maintained 
it  is  well  to  use  thermometers  with  particularly  wide  divisions  on  the  scale 
(1°  C.  =  1  cm.).     These  thermometers  need  only  embrace  a  moderate  range  of 
degrees  (about  40°-80°  or  45°-85°).      They  can  be  obtained  from  A.  Haak 
in  Jena. 


340  COLLECTED  STUDIES  IN  IMMUNITY. 

For  many  years,  owing  to  the  valuable  researches  of  Buchner, 
an  inactivation  by  means  of  temperature  of  55-56°  was  regarded 
as  practically  a  specific  criterion  for  the  alexins.  We  now  know, 
however,  that  no  general  rule  can  be  formulated  in  this  respect.  On 
the  one  hand  there  are  complements  which  are  not  at  all  influenced  by 
the  customary  half-hour's  heating  to  55°  C.  (thermostable  comple- 
ments), and  on  the  other  there  are  amboceptors  which  are  com- 
pletely destroyed  by  such  heating.  A  complement  belonging  to  the 
first  category  was  first  described  by  Ehrlich  and  Morgenroth 1  as 
occurring  in  considerable  amount  in  normal  goat  serum  and  in  the 
serum  of  a  buck  which  had  been  immunized  with  sheep  serum;  and 
thermolabile  amboceptors,  especially  in  normal  sera,  are  not  at  all 
rare.  Thus  the  amboceptor  above  mentioned  regularly  present  in 
horse  serum  and  acting  on  guinea-pig  blood,  as  well  as  one  studied 
by  Sachs  2  present  in  dog  serum  and  also  acting  on  guinea-pig  blood, 
is  completely  destroyed  by  half  an  hour's  heating  to  55°  C.  Hence 
the  first  rule  in  the  demonstration  of  the  complex  character  of  hsemo- 
lytic  poisons  by  thermogenic  inactivation  is  always  to  employ  the 
lowest  temperature  at  which  inactivation  takes  place  within  a  short 
time  (20-60  minutes).3 

VI.   The    Quantitative    Estimation    of   Amboceptors,  Complements 

and  Receptors. 

In  special  cases,  e.g.  during  the  course  of  an  immunization, 
it  is  of  considerable  value  to  accurately  determine  the  amounts  of 
amboceptor  and  complement  present  in  the  serum.  While  referring 
to  the  studies  of  v.  Dungern  (p.  36),  Bulloch  (I.e.),  Morgenroth  and 
Sachs  (pp.  226  and  250),  we  should  like  to  emphasize  that,  in  general, 
in  determining  the  amount  of  complement  it  is  necessary  to  make 

1  See  page  13. 

2  See  page  181  et  seq. 

3  According  to  the  researches  of  Korschun  and  Morgenroth    (see  pp.  267 
et  seq.)  the  hsemolytic  substances  of  organ  extracts  are  "coctostable,"  i.e., 
they  are  not  destroyed  even  by  several  hours'  boiling.     Hence  we  designate  a 
substance  as 

Thermolabik,  if  it  is  rendered  inert  by  heating  to  55°-56°  C.; 

Thermostable,  if  it  withstands  heating  to  56°  or  over  but  is  destroyed  by 

boiling; 

Coctostable,  if  it  resists  boiling  at  100°  C. 

In  special  cases  in  order  to  still  more  closely  .characterize  their  behavior 
jone  can  add  temperature  and  duration  of  heat  as  an  index. 


METHODS  OF  STUDYING  H^MOLYSINS.  341 

two  determinations,  namely,  one  carried  out  with  the  single-solvent 
dose  of  amboceptor,  the  other  with  a  high  multiple  of  the  same. 
The  reasons  for  this  procedure  can  be  found  in  the  study  on  the 
quantitative  estimation  of  amboceptor,  complement,  and  anticom- 
plement  (page  250). 

So  far  as  the  estimation  of  the  amount  of  amboceptor  is  concerned, 
this  is  effected  according  to  similar  principles,  and  usually  in  such  a 
way  that  one  works  with  an  excess  of  complement.  A  certain  diffi- 
culty is  encountered  in  the  fact  that  the  amount  of  complement 
contained  in  the  serum,  e.g.,  rabbit  serum,  is  variable.  It  is  there- 
fore always  necessary,  in  order  to  exclude  this  disturbing  factor,  to 
first  determine  the  activating  value  of  the  complementing  serum 
using  a  specimen  of  the  immune  serum  in  question  as  a  standard 
serum.  Directly  after  this  test  by  which  the  amount  of  complement 
is  strictly  defined,  the  quantitative  estimation  of  amboceptor  in 
the  new  serum  must  be  undertaken. 

It  is  also  important  to  estimate  the  amount  of  receptor  present 
in  the  red  blood-cells:  the  measure  of  this  is  the  binding  of  ambo- 
ceptor. 

Erhlich  and  Morgenroth  (see  pages  72  et  seq.)  have  demon- 
strated that  the  binding  capacity  of  red  blood-cells  varies  to  an 
extraordinary  degree.  While  in  many  combinations  the  blood-cells 
combine  with  just  that  amount  of  amboceptor,  which  on  the  addi- 
tion of  suitable  complement  leads  to  their  complete  solution  (ambo- 
ceptor unit},  it  was  found  that  in  numerous  other  cases  the  blood- 
cells  are  able  to  take  up  as  high  as  100  single-solvent  doses  of  ambo- 
ceptor. Corresponding  to  the  amboceptor  unit,  the  receptor  unit, 
is  that  amount  of  receptor  which  combines  with  one  amboceptor 
unit  (see  page  254).  The  combining  power  of  the  erythrocytes  is 
determined  by  adding  varying  multiples  of  the  amboceptor  unit 
to  the  blood-cells,  centrifuging  at  the  end  of  about  an  hour  and  then 
allowing  the  various  decanted  fluids  to  act  on  fresh  blood-cells  in 
the  presence  of  sufficient  complement.  The  degree  of  haemolysis 
which  occurs  readily  shows  just  how  much  amboceptor  was  still 
completely  bound.  (See  page  75  and  the  protocols  on  pages  98 
and  99.)1 

1  Concerning  the  extraordinarily  large  binding  capacity  of  bacteria  for  agglu- 
tinins  and  for  amboceptors,  see  the  interesting  communication  of  Eisenberg 
and  Volk  (Zeitsch.  f.  Hygiene,  Vol.  80)  and  of  Pfeiffer  and  Friedberger  (Berl. 
klin.  Wochensch.  1902,  No.  25.) 


342  COLLECTED  STUDIES  IN   IMMUNITY. 

Finally,  in  studying  the  complements  of  a  serum  it  is  often  of 
considerable  importance  to  determine  their  plurality.  The  methods 
leading  to  a  differentiation  of  the  separate  complements  have  been 
described  in  detail  in  a  number  of  places,  so  that  we  can  here  con- 
tent ourselves  by  referring  to  the  studies  of  Ehrlich  and  Morgenroth 
(pages  11-56,  110),  of  Ehrlich  and  Sachs  (page  195),  and  of  Marshall 
and  Morgenroth  (page  222). 

VII.  The  Study  of  Antihaemolytic  Actions. 

The  subject  of  antihsemolytic  functions,  which  has  only  recently 
been  carefully  worked  up,  has  attained  considerable  importance 
for  the  comprehension  of  the  mechanism  of  hsemolysins.  Although 
at  the  present  time  the  study  of  the  influences  inhibiting  haemolysis 
is  not  at  all  complete,  it  is  possible  at  least  to  indicate  certain  gen- 
eral principles. 

We  shall  begin  with  the  simple  hsemotoxins,  which  are  character- 
ized by  a  cytophile  haptophore  group  and  a  zymotoxic  group. 
(Analogous  to  these  are  the  hsemagglutinins,  also  characterized  by 
a  cytophile  haptophore  group  and  an  agglutinating  group.)  If  we 
analyze  the  action  of  these  hsemotoxins,  we  see  that  this  can  be  inhib- 
ited in  two  ways: 

(1)  By  means  of  an  antibody  which  fits  into  the  haptophore 
group  and  -so  deflects  this  from  the  receptor  of  the  cell. 

(2)  By   means   of   substances  which   are   capable   of  occupying 
the  receptor  of  the  blood-cell  and  so  block  this  for  the  entrance  of 
the  hsemo toxin. 

So  far  as  the  first  group  is  concerned,  such  antibodies  are  well 
known  for  a  large  number  of  blood  poisons.  We  need  only  call  to 
mind  the  antihsemolysins,  such  as  anticrotin,  antitetanolysin,  anti- 
staphylolysin,  antibodies  against  the  hsemolytic  venoms  of  snakes, 
spiders,  and  toads.  Besides  these  there  are  the  antiagglutinins,  such 
as  antiricin,  antiabrin,  anticrotin.  These  substances  can  be  produced 
as  antitoxins  by  means  of  immunization,  but  they  also  occur  in 
normal  serum,  as,  for  example,  antitetanolysin  in  horse  serum  (Ehrlich), 
antistaphylolysin  in  serum  from  goats,  man,  and  horse  (M.  Neisser 
and  Wechsberg). 

The  second  method  of  inhibition  is  effected  by  substances  which 
occupy  the  receptors  of  the  cells.  Hence  these  must  be  substances 
which  possess  the  same  haptophore  group  as  the  hsemotoxins  them- 


METHODS  OF  STUDYING  ELEMOLYSINS.  343 

selves.  This,  however,  at  once  leads  to  the  idea  that  transformation 
products  of  the  hsemolysin  itself  could  exert  this  action.  Ehrlich 's 
researches  on  the  constitution  of  diphtheria  poison  have  shown  that 
in  toxins  and  related  bodies  the  zymotoxic  group  is  far  less  stable 
than  the  haptophore  group.  The  bodies  so  derived,  toxoids,  still 
possess  the  property  of  combining  with  the  cell  receptors,  they  are 
still  able  to  neutralize  antitoxin,  and  to  excite  the  reactive  formation 
of  antibodies,  but  they  more  or  less  completely  lack  any  toxicity. 
This  formation  of  toxoid,  first  described  by  Ehrlich,  has  since  been 
demonstrated  for  a  number  of  substances,  hsemotoxins  (tetanolysin, 
snake  venom,  staphylolysin) ,  as  well  as  agglutinins  and  coagulins.1 
Ehrlich  in  his  first  study  already  pointed  out  that  an  increase  in 
the  haptophore 's  affinity,  developing  in  the  course  of  toxoid  for- 
mation, was  conceivable.  The  toxoid  which  was  thus  produced 
would  then  be  able,  owing  to  the  increased  affinity,  to  unite  with 
the  receptor  of  the  cell  even  in  competition  with  the  unchanged 
toxin.  In  this  way  the  toxoid  would  protect  the  cell  against  the 
entrance  of  the  real  poison,  and  of  course,  against  the  poison's  injurious 
influence.  For  these  toxoids  Ehrlich  has  proposed  the  term  pro- 
toxoids.  Of  course  such  a  protective  effect  can  also  be  produced 
in  conformity  with  the  laws  of  mass  action  by  toxoids  having  the 
same  affinity  (syntoxoid)  to  the  cell  receptor  as  the  toxin,  whereas 
the  protection  will  be  slight  or  minimal  if,  as  a  result  of  toxoid  for- 
mation, there  is  a  decrease  in  the  haptophore  group's  affinity  (epi- 
toxoid).  Recent  investigations  on  the  agglutinins  of  bacteria2  and 
on  coagulins  have  shown  that  by  heating  these  substances,  agglu- 
tinoids,  which  possess  a  higher  affinity  than  the  agglutinins  them- 
selves, are  developed  in  considerable  quantities.  These  are,  there- 
fore, termed  proagglutinoids. 

It  is  an  easy  matter  in  any  given  instance  to  determine  experi- 
mentally which  of  these  two  inhibitory  processes  is  present.  If 
one  is  dealing  with  a  certain  particular  serum  which  inhibits  the 
action  of  the  hsemotoxin,  it  may  be  regarded  a  priori  as  probable 
that  the  substance  in  question  is  an  antibody  in  the  ordinary  sense. 
This  becomes  almost  certain  if  the  serum  was  derived  from  an  animal 
specifically  immunized.  Experimentally  it  is  easy  to  show  that 


1  Eisenberg  and  Volk,  Zeitsch.  f.  Hygiene,  Vol.  40,  1902;    Bail,    Archiv 
f.  Hygiene,  Vol.  42,  1902;  Shiga,  page  312. 

2  Ibid. 


344  COLLECTED  STUDIES  IX  IMMUNITY. 

the  antibody  belongs  to  this  group  as  follows:  The  red  blood-cells 
are  treated  with  a  just  neutral  mixture  of  haemotoxin  and  antibody 
and  then  centrifuged.  If  there  was  a  true  deflection  of  the  poison, 
these  cells  must  now  behave  exactly  like  fresh  blood-cells;  above 
all  they  must  still  possess  exactly  the  original  binding  capacity  for 
the  hsemo toxin. 

In  contrast  to  this  behavior,  the  disturbance  caused  by  trans- 
formation products  of  the  hsemolysin  itself  manifests  itself  even 
in  experiments  made  only  with  blood-cells  and  the  toxic  substance. 
The  experimental  series  has  an  irregular  course  analogous  to  Shiga's 
experiments  with  agglutinins.  For  example,  if  increasing  amounts 
of  agglutinating  serum  which  has  previously  been  heated  are  added 
to  dysentery  bacilli,  one  can  observe  that  the  test-tubes  containing 
the  largest  amount  of  agglutinins  show  no  agglutination;  and  that 
agglutination  shows  itself  only  in  the  tubes  containing  smaller  amounts 
and  disappears  again  with  still  smaller  quantities. 

In  order  to  show  that  in  this  case  there  is  no  real  occupation 
of  the  receptors  by  the  proagglutinoid,  one  tests  the  behavior  of  the 
centrifuged  bacteria.  These  are  suspended  in  salt  solution,  and 
again  mixed  with  what  is  otherwise  an  effective  dose  of  agglutinin. 
They  are  no  longer  agglutinated  because  the  agglutinin  cannot  com- 
bine with  the  blocked  receptors.  We  do  not  doubt  at  all  that  this 
phenomenon  will  also  be  found  in  haemagglutinins. 

The  conditions  are  far  more  complicated  with  the  complex  hae- 
molysins,  the  possibilities  for  the  inhibitory  mechanism  being  more 
numerous.  It  may,  therefore,  be  well  to  aid  our  analysis  by  means 
of  a  diagram  (see  opposite). 

The  diagram  refers  to  experiments  made  with  mixtures  which 
do  not  by  themselves  dissolve  blood-cells,  and  whose  composition 
must  first  be  accurately  determined  quantitatively.  One  next  devises 
a  hsemolytic  combination  in  which  amboceptor  and  complement  are 
present  in  exact  equivalence  and  determines  the  amount  of  the  anti- 
body in  question  which  will  just  inhibit  the  action  of  this  combina- 
tion. By  means  of  this  exactly  balanced  mixture  experiments  by 
the  centrifuge  method  are  made  both  with  the  sediments  and 
with  the  decanted  portions  as  shown  in  the  diagram.1 

1  This  method  refers  to  cases  I,  III,  and  IV  of  the  scheme,  while  case  II 
refers  to  an  experiment  made  with  ordinary  complementoid  serum  obtained  by 
heating. 


METHODS  OF  STUDYING  H^MOLYSINS. 


345 


1 

Behavior  of 

Behavior  of  the 

the  Sediment. 

Decanted  Fluid. 

I:i 

u    ' 

ll 

1 

1 

|o^ 

M§o 

c| 

I 

c 

1^° 

ft°t 

1-2 

fa 

•M 

111 

'%A^  \ 

1° 

Js 

Soo 

< 

o 

o 

c-^J          Anticomplement. 

ac—  tef      c'  complement; 
x3p/       ac,  anticomplement. 

0 

0 

0 

n-                     at 

fmm    ^Blocking  of  the  com-      0° 
plementophile 
C^-M          group  of  the  am- 
0\               boceptor    (a)    by 
1          means  of  ocmple-      at 
|y\)         mentoid  cd.              37° 

0 

0 
0 

0 
0 

0 
0 

a 

JIII. 

antiamboceptor  (aa)  . 
a,  amboceptor. 

0 

+ 

0 

+ 

§cytophilic    protoam- 
boceptoid  (ca). 
receptor   of  the  red 
blood-cell,  r. 

0 

0 

- 

+ 

346  COLLECTED  STUDIES  IN  IMMUNITY. 

In  studying  the  sediments  the  question  is  always  whether  these 
have  taken  up  amboceptor  or  not.  This  is  most  easily  determined 
by  the  addition  of  complement.  This  procedure,  however,  should 
be  supplemented  by  the  more  difficult  and  troublesome  investigation 
of  the  binding  power  of  the  blood-cells  for  newly  added  amboceptor. 
In  this  case,  of  course,  a  parallel  test  with  untreated  blood-cells  fur- 
nishes the  basis  for  comparison.  As  a  rule  experiments  at  moderate 
temperatures  suffice;  only  hi  case  II  is  a  variation  of  temperature 
required. 

In  case  I  the  complement  is  deflected  by  means  of  an  anticomple- 
ment.  One  must  take  into  consideration  both  natural  anticom- 
plements  and  those  artificially  produced  by  immunization;  further- 
more, attention  must  be  paid  to  similarly  acting  derivatives  of  the 
amboceptors,  the  amboceptors,  whose  complementophile  group  has 
been  preserved.1  The  behavior  of  the  amboceptoids,  especially  in 
those  cases  in  which  the  affinity  of  the  complementophile  group  of 
these  amboceptoids  has  become  increased,  will  in  no  way  differ  from 
that  of  the  anticomplements.  Finally  we  must  remember  that  the 
.amboceptors  can  act  in  a  way  like  anticomplements  as  a  result  of 
the  deflection  of  complements  by  excess  of  amboceptor,  a  phenomenon 
first  described  by  Neisser  and  Wechsberg  (see  page  120).  In  that 
case,  of  course,  the  decanted  fluid  contains  the  excess  of  amboceptors 
.and  the  complement  bound  to  the  same. 

II.  In  case  II  the  complementophile  group  of  the  amboceptor  is 
blocked.     Here  we  must  first  consider  the  action  of  complementoids 
(see  Ehrlich  and  Sachs,  page  209) ,  although,  according  to  our  present 
experiences,  these  only  seldom  come  into  play  because,  in  the  forma- 
tion of  complementoid,  there  is  usually  a  decrease  of  affinity. 

III.  The  third  possibility  is  the  action  of  antiamboceptors  which 
fit  into   the  cytophile  group  of  the  amboceptors.      They  may  be 
present  normally  or  produced  artificially  by  immunization.     From 
a  theoretical  standpoint  these  antiamboceptors  are  to  be  identified 
with  the  receptors  of  the  cells  into  which  the  amboceptors  fit.     Hence 
thrust-off  receptors  present  in  solution  will  act  as  antiamboceptors.2 
According  to  recent  investigations  the  serum  against  snake  venom 


1  Wechsberg,  Wiener  klin.  Wochensch.  1902,  No.  28;  E.  Neisser  and  Friede- 
mann,  Berl.  klin.  Wochenschr.  1902,  No.  29. 

'Morgenroth,  page  241;  also  P.  Miiller,  Munch,  med.  Wochenschr.  1902, 
No.  32. 


METHODS  OF  STUDYING  H^MOLYSIXS.  347 

\ 
also  contains  antiamboceptors  against  the  amboceptors  of  cobra  venom. 

IV.  The  fourth  possibility  consists  in  the  occupation  of  the  recep- 
tor by  cytophile  proamboceptoids,  conditions  which  correspond  to 
those  discussed  under  the  simple  hasmotoxins  (page  342).  Since  the 
study  of  amboceptoids  is  still  in  its  infancy,  such  cases  have  not  yet 
been  described.  Their  occurrence,  however,  is  extremely  probable 
and  the  near  future  will  probably  furnish  experiences  in  this  direction. 

So  far  as  the  details  of  the  experiments  are  concerned,  the  previous 
papers  furnish  detailed  descriptions  which  may  be  consulted.  The 
reader  is  referred  to  the  following:  Case  I,  pages  224-259;  II,  page 
209;  III,  pages  103,  104,  248. 

In  any  particular  instance  it  is  necessary  to  determine  which 
of  these  cases  obtains.  Above  all  it  is  necessary  to  remember  that 
the  inhibition  need  not  always  be  due  to  a  specific  binding,  but  that 
it  may  be  caused  by  disturbing  factors,  which  we  have  classed  to- 
gether under  the  term  antireactive  actions. 

For  example,  if  the  union  of  amboceptor  and  complement  does 
not  take  place  at  low  temperatures,  or  if  owing  to  the  action  of  salt 
the  union  of  complement  with  the  anchored  amboceptor,  or  of  ambo- 
ceptor to  the  cell  receptor,  is  hindered,  these  are  the  result  of  anti- 
reactive  influences  and  not  of  specific  inhibitions.  As  a  rule  it  is  easy  in 
any  given  case  to  decide  which  kind  of  inhibition  is  present.  In  most 
cases  the  origin  and  mode  of  derivation  of  the  substances  in  question 
give  valuable  clues  in  this  direction.  If  antireactive  influences  can  be 
excluded,  it  is  not  difficult  by  a  logical  application  of  the  centrifugal 
method  to  classify  the  case  under  one  of  the  heads  given  in  the  table. 

Naturally  these  cases  may  also  be  combined.  Thus,  for  example, 
a  fluid  may  contain  simultaneously  anticomplement,  antiamboceptor 
or  anticomplement  and  procomplementoid.  Antiamboceptor  and 
amboceptor,  complement  and  anticomplement  in  one  solution  can  be 
excluded,  since  they  mutually  neutralize  each  other. 

Another  important  point  which  belongs  here  is  the  recognition 
of  concealed  amboceptors,  whose  activatibility  is  suppressed  by  the 
simultaneous  presence  of  anticomplement.  For  the  experimental 
technique  see  Morgenroth,  page  245. 

It  is,  of  course,  impossible  to  treat  exhaustively  aU  the  innumer- 
able variations  which  come  into  question.  We  hope,  however,  that 
the  methodical  exposition  here  given  has  shown  how  the  fundamental 
doctrines  of  Ehrlich's  Side-chain  Theory  make  a  systematic  study  of 
hsemolysins  possible. 


XXX.  THE   TECHNIQUE   OF  BACTERICIDAL   TEST- 
TUBE   EXPERIMENTS. 

By  Professor  M.  NEISSER,  Member  of  the  Institute. 

IN  order  to  measure  the  bactericidal  power  of  a  serum  or  of 
serum  mixture  by  means  of  a  test-tube  experiment,  the  plate  method 
(Neisser,  Buchner)  is  still  the  safest.  Only  in  special  cases  can  one 
obtain  useful  comparative  results  by  other  methods  (observing 
hanging  drops  for  the  onset  of  granular  degeneration,  R.  Pfeiffer, 
or  bioscopic  method,  M.  Neisser  and  Wechsberg *).  But  even  the  plate 
method  at  present  is  cumbersome  and,  what  is  of  more  consequence, 
is  not  applicable  in  all  cases.  It  is  not  a  sensitive  method  and  is  only 
then  useful  when  marked  results  are  to  be  expected  in  consequence 
of  strong  bactericidal  powers.  As  a  rule  such  marked  results  are 
only  to  be  attained  with  immune  sera  and  only  rarely  with  normal 
sera. 

So  far  as  immunization  is  concerned  it  is  impossible  to  make 
general  statements,  and  I  shall  therefore  only  cite  a  few  examples. 
Thus  in  the  case  of  chlorea  vibrios  a  single  subcutaneous  injection 
of  three  dead  agar  cultures  into  rabbits  gives  good  results  (R.  Pfeiffer 
and  Marx2),  as  does  also  the  intravenous  injection  of  extremely  small 
quantities  (Mertens,  R.  Pfeiffer3).  In  immunizing  against  typhoid, 
dogs  and  goats  are  most  useful.  In  this  case  a  single  injection  of 
dead  cultures  does  not  suffice  in  order  to  obtain  a  high-grade  bac- 
tericidal serum;  on  the  contrary  repeated  injections  of  living  cul- 
tures are  necessary.  For  obtaining  a  serum  having  strong  bac- 
tericidal properties  against  Shiga's  dysentery  bacilli,  horses  are  well 
adapted;  goats  very  much  less  so;  rabbits  and  guinea-pigs  are  very 

1  Munch,  med.  Wochensch.  1900,  No.  37. 

2  Zeitschr.  f .  Hygiene,  XXVII,  1898. 

3  Deutsche  med.  Wochensch.  1901. 

348 


TECHNIQUE  OF  BACTERICIDAL  TEST-TUBE  EXPERIMENTS.  349 

ill-suited  for  this  purpose.  One  should,  of  course,  never  forget 
to  examine  the  normal  serum  for  bactericidal  powers  previous  to 
immunization. 

With  a  great  many  bacteria  it  has  not  yet  been  possible  to  pro- 
duce a  serum  bactericidal  in  vitro.  Thus  our  experiments  in  this 
direction  extending  over  many  years  were  unsuccessful  with  staphy- 
lococcus  pyogenes  aureus  (goat,  rabbit)  and  with  the  diphtheria 
bacillus.  Nor  have  we  been  able  thus  far  to  obtain  bactericidal 
effects  hi  vitro  from  Susserin  and  other  similar  sera  which  are  effective 
in  animal  tests.  The  reasons  for  this  behavior  are  not  yet  clear, 
and  they  are  therefore  still  being  studied. 

Bordet  and  Gengou  have  devised  a  method  (Annales  de  ITnstitut 
Pasteur  1901)  by  the  aid  of  which  a  bactericidal  interbody  pro- 
duced by  immunization  can  be  recognized  even  in  those  cases  in 
which  plate  experiments  fail  (e.g.  erysipelas  of  swine).  This  method 
depends  on  the  property,  said  to  be  possessed  by  bacteria  to  which 
interbody  has  been  supplied,  of  combining  also  with  hsemolytic  com- 
plements. This  loss  of  complement,  which  can  be  readily  detected, 
shows  that  the  bacteria  have  combined  with  a  bactericidal  inter- 
body. Without  entering  into  the  theoretical  significance  of  this 
interesting  experiment  we  shall  content  ourselves  by  saying  that 
in  several  cases  in  which  we  tested  bactericidal  immune  sera  in  this 
way  we  failed  to  obtain  satisfactory  results.  The  method  does  not 
seem  to  us  to  be  suited  to  a  quantitative  estimation  of  an  immune 
serum. 

It  need  hardly  be  said  that  the  first  requisite  for  the  success  of 
bactericidal  experiments  is  that  all  vessels,  diluting  fluids,  as  well 
as  the  sera  employed  be  absolutely  sterile.  Great  care  is  necessary, 
especially  in  collecting  the  blood.  The  method  described  in  the 
preceding  chapter  for  bleeding  rabbits  and  guinea-pigs  is  sufficient 
to  obtain  sterile  blood.  For  collecting  smaller  quantities  of  blood 
from  the  ear  vein  of  rabbits  it  is  necessary  to  first  cleanse  the  ear 
with  70%  alcohol  and  then  thrusting  a  short  sterile  hollow  needle 
into  a  vein.  In  many  cases,  to  be  sure,  the  blood  can  also  be  col- 
lected by  making  a  short  incision  across  the  marginal  ear  vein  with 
a  sterile  scalpel,  and  then,  by  holding  the  animal  properly,  allowing 
the  blood  to  flow  out  without  running  over  the  ear. 

In  bleeding  pigeons  and  chickens  by  decapitation  one  cannot 
always  count  on  sterile  serum;  hence  it  is  well  to  lay  bare  the  vessels 
of  the  neck.  For  repeated  bleeding  of  guinea-pigs  one  must  also 


350  COLLECTED  STUDIES  IN  IMMUNITY. 

collect  the  blood  directly  from  the  vessels  of  the  neck  and  then  tie 
the  vessel.  It  is  an  easy  matter  to  obtain  very  small  quantities 
of  sterile  pigeon  blood  from  the  wing  veins  by  first  carefully  removing 
the  feathers,  disinfecting  the  skin  with  alcohol  and  then  after  incising, 
touching  the  skin  as  little  as  possible. 

For  purposes  of  collecting  the  serum,  the  blood  is  either  allowed 
to  stand  overnight  (see  the  preceding  chapter),  or  by  means  of  a 
sterile  funnel  is  allowed  to  flow  into  a  sterile  bottle  containing  sterile 
glass  beads  or  steel  shavings.  The  bottle  is  then  stoppered  with 
a  cork  (previously  burnt  off),  the  blood  defibrinated  by  shaking, 
and  then  centrifuged.  As  a  rule,  centrifuging  does  not  injure  the 
serum,  especially  if  afterwards  the  upper  layer  of  fluid  is  siphoned 
off.  For  absolutely  certain  sterility  the  spontaneous  separation 
of  the  serum  is  to  be  preferred  to  defibrination  and  centrifuging. 

The  active  sera  used  for  complementing  are  to  be  employed  as 
fresh  as  possible,  in  no  case  more  than  two  or  three  days  old  (refriger- 
ator). The  immune  sera,  which  are  usually  employed  in  the  inactive 
state,  will  keep  in  the  refrigerator  for  a  long  time.  Even  in  these, 
however,  a  loss  of  power  is  observed.  In  the  case  of  high-grade 
immune  sera  the  addition  of  0.5%  phenol  is  allowable  for  preserva- 
tion. In  the  small  quantities  in  which  the  serum  is  used  in  experi- 
ments (about  0.01  cc.)  this  amount  of  phenol  is  without  effect  either 
on  the  bacteria  or  on  the  complements. 

Before  commencing  the  experiment  proper  it  is  necessary  to 
determine  what  amount  sown  gives  the  most  favorable  results. 
Thus  in  many  experiments  it  may  be  of  advantage  to  always  sow 
Vsoo  cc.  of  a  one-day  bouillon  culture,  whereas  with  another  bac- 
terium sowing  Viooo  or  Vioooo  l^op  of  a  one-day  agar  culture  will 
give  more  uniform  results.  It  is  further  necessary  to  repeatedly 
convince  one's  self  that  the  control  plates  regularly  show  a  uniformly 
good  growth,  for  only  when  that  is  the  case  can  uniform  results  be 
expected.  For  example,  although  the  bacillus  of  hog  cholera  grows 
very  well  on  ordinary  slant  agar,  the  control  plates  may  result 
most  irregularly.  In  that  case  one  can  make  use  of  glycerine  agar. 
Other  bacteria  again  do  not  bear  suspension  in  0.85%  salt  solution 
at  all  well;  in  that  case  one  must  use  bouillon  cultures  and  make 
the  dilutions  with  bouillon  instead  of  with  salt  solution.  The  dilu- 
tion should  always  be  managed  so  that  the  amount  finally  sown  is 
about  5-10  drops,  for  in  sowing  only  1  or  2  drops  considerable  varia- 
tions in  the  number  of  colonies  may  occur.  In  any  case,  however,  the 


TECHNIQUE  OF  BACTERICIDAL  TEST-TUBE  EXPERIMENTS.  351 

plate  sown  must  contain  many  thousands  or  an  innumerable  number 
of  colonies.  The  bactericidal  effect  will  then  be  distinctly  shown  by 
the  reduction  in  the  proper  plates  of  this  large  number  of  colonies 
to  zero  or  almost  zero. 

The  test-tubes  most  advantageously  employed  are  the  little 
tubes  9-10  cm.  long  and  1.3  cm.  diameter.  The  cotton  stoppers  are 
removed  and  all  the  different  components  filled  into  the  tubes.  Then 
the  stoppers  are  replaced  after  being  flamed.  If  the  air  is  at  all 
still  one  need  not  fear  keeping  the  tubes  open  for  this  length  of  time. 

In  testing  an  immune  serum  one  commences  by  examining  the 
immune  serum  in  the  fresh  active  state,  and,  of  course,  in  the  same 
manner  that  the  serum  of  the  animal  in  question  was  examined  pre- 
vious to  immunization.  For  this  purpose  a  number  of  test-tubes  are 
filled  with  1.0,  0.3,  0.1,  0.03,  0.01  cc.  of  the  fresh  active  serum.  Finer 
gradations  are  useless  in  view  of  the  lack  of  sensitiveness  of  the  test- 
tube  method.  This  we  have  already  pointed  out.  The  amount  of 
culture  to  be  sown  is  then  added  and  all  tubes  filled  up  to  2  cc.  with 
physiological  salt  solution.  Finally  three  drops  of  bouillon  are  added 
to  each  tube.  The  addition  of  bouillon  has  proven  to  be  of  consider- 
able value,  for  it  suffices  to  balance  disturbing  variations  of  the 
osmotic  pressure.  It  is  important  to  make  the  total  volume  of  fluid 
the  same  in  all  the  tubes  by  the  addition  of  fluid.  Besides  this  it  is 
important  to  have  a  number  of  controls,  namely,  a  control  of  the 
culture  sown,  second,  a  control  testing  the  sterility  of  the  maximum 
amount  of  serum  employed,  and  third,  a  control,  or  better  a  series 
of  controls,  containing  the  culture  sown  plus  the  serum  in  an  inactive 
form.  By  means  of  this  last  control  one  can  see  whether  a  thermostable 
complement  is  present  or  not.  It  also  serves  to  show  that  the  bacteri- 
cidal action  is  not  simulated  by  the  agglutinating  power  of  the  serum. 

The  tubes  are  now  kept  in  the  thermostat  for  at  least  three  hours, 
having  previously,  however,  been  carefully  shaken.  On  being  taken 
out  of  the  thermostat  they  are  again  carefully  shaken  and  then 
worked  up  into  plates.  For  this  purpose  5-10  drops  are  taken  from 
each  tube  by  means  of  uniform  pipettes  and  made  into  plates  in  the 
usual  way.  The  plates  are  placed  in  the  thermostat  upside  down, 
and  kept  there  until  the  following  day.  The  growth  is  best  and 
most  rapidly  described  by  means  of  approximate  estimates,  using  a 
scheme  somewhat  as  follows:  0  or  almost  0,  about  100,  several  hun- 
dreds, thousands,  very  many  thousands,  infinite  number.  A  distinct 
bactericidal  action  is  only  then  present  if  the  controls  result  as  they 


352  COLLECTED  STUDIES  IN  IMMUNITY. 

should,  and  if  a  reduction  of  colonies  from  an  infinite  number  or 
many  thousands  to  0  or  very  few  has  occurred.  Furthermore  the 
test  can  only  then  be  regarded  as  having  a  good  result  if  the  lower 
limits  of  the  amount  of  active  serum  have  been  reached,  i.e.,  when 
the  last  plates  again  show  an  increase  in  the  number  of  colonies. 

A  certain  degree  of  control  on  the  plate  experiments  is  obtained 
in  suitable  cases  by  placing  the  tubes  (from  which  a  few  drops  were 
taken  for  sowing  into  plates)  into  a  thermostat  and  observing  them 
the  next  day.  In  this  case  the  culture  controls  show  a  luxuriant 
growth,  while  in  the  other  test-tubes,  depending  on  the  amount  of 
serum,  either  a  growth  will  occur  or  not.  This  test-tube  experi- 
ment, of  course,  will  only  then  show  a  result  if  the  bactericidal 
power  of  the  serum  was  large  enough  to  kill  even  the  last  germ 
in  the  corresponding  specimens.  But  if  even  only  a  few  germs 
remain  alive  (in  consequence,  for  example,  of  a  special  resistance), 
it  will  be  found  that  these  few,  after  the  bactericidal  substances 
are  used  up,  will  again  multiply  enormously.  Hence  the  test-tube 
method  cannot  give  reliable  results  in  spore-bearing  bacteria. 
For  the  same  reason  it  is  important,  in  making  plate  tests,  to 
keep  the  tubes  in  the  thermostat  for  a  certain  particular  time, 
which  must  be  determined  separately  for  each  bacterium;  for  it 
must  be  borne  in  mind  that  the  killing  of  the  bacteria  can  be 
represented  by  a  curve  whose  lowest  point  (lowest  number  of 
living  germs)  must  be  approximately  attained  if  marked  results  are 
desired.  Either  side  of  this  point,  unless  this  point  be  0,  the  results 
will  be  correspondingly  less.  Smaller  results,  however,  are  worth- 
less for  all  these  experiments,  as  is  seen  when  we  consider  that  agglu- 
tination, although  it  has  so  little  directly  to  do  with  bactericidal 
action,  is  also  able  to  cause  a  decrease  in  the  number  of  colonies  on 
a  plate  and  thus  simulate  a  decrease  in  the  number  of  germs.  This 
is  one  of  the  reasons  why  the  control  described  above  with  inactivated 
serum,  in  which,  of  course,  the  agglutinin  is  still  present,  is  so  im- 
portant. 

After  the  fresh  active  immune  serum  has  been  tested  as  to  its 
bactericidal  power  one  proceeds  with  the  examination  of  the  inactive 
immune  serum  plus  complement.  Inactivation  is  accomplished  in 
accordance  with  the  principles  laid  down  in  the  preceding  chapter. 
For  complement  one  chooses  first  the  normal  serum  of  the  species 
from  which  the  immune  serum  is  derived.  A  preliminary  trial  will 
then  be  necessary  to  show  what  dose  of  this  normal  serum  can  be 


TECHNIQUE  OF  BACTERICIDAL  TEST-TUBE  EXPERIMENTS.  353 

employed  without  causing  bactericidal  action  by  the  normal  serum 
itself. 

The  dose  of  complement  should  be  such  that  the  plate  containing 
only  complement  and  the  culture  differs  very  little  from  the  control 
of  the  culture  sowing  alone.  Too  large  a  quantity  of  complement 
should  be  avoided;  certainly  in  no  case  should  more  than  about  0.5  cc. 
complementing  serum  be  used.  The  technique  then  is  as  follows: 
1.0,  0.3,  0.1,  0.03,  0.01  cc.  of  inactive  immune  serum  are  placed  into 
a  series  of  test-tubes;  to  each  of  these  is  then  added  the  same  amount 
of  the  complementing  active  normal  serum  (e.g.  0.3  cc.)  and  the 
bacterial  culture.  All  of  the  tubes  are  then  made  up  to  the  same 
amount  (2  to  3  cc.)  with  physiological  salt  solution,  and  finally  each 
tube  receives  three  drops  of  bouillon.  The  controls  in  this  case 
must  be  still  more  numerous.  The  sterility  of  each  serum  must  be 
demonstrated,  as  well  as  the  fact  that  the  inactive  immune  serum  by 
itself  and  the  active  normal  serum  by  itself  are  inert. 

The  result  of  such  an  experiment  is  usually  startling  at  first  sight 
because  the  plates  which  had  the  largest  amounts  of  immune  serum 
show  the  largest  number  of  colonies.  One  must  therefore  always 
bear  in  mind  the  deflection  of  complements  in  consequence  of  an 
excess  of  immune  body.  The  paradoxical  results  caused  by  this 
deflection  of  complement  is  seen  not  only  in  the  plates  but  also  in 
the  test-tube  experiment.  The  various  ways  in  which  the  comple- 
ment is  deflected  from  its  destination  have  already  been  discussed 
in  a  previous  chapter.  In  bactericidal  experiments  the  deflection 
caused  by  an  excess  of  the  amboceptors  produced  by  immunization 
is  especially  important.  In  a  mixture  of  bacteria,  complements,  and 
large  amounts  of  amboceptor,  the  complement  is  bound  not  only  by 
the  amboceptors  anchored  to  the  bacteria  but  also  in  large  measure 
by  "  free  "  amboceptors  which  are  not  anchored  to  bacteria.  A 
portion  of  the  anchored  amboceptor  therefore  finds  no  complement 
at  its  disposal  and  is,  therefore,  unable  to  exert  any  bactericidal 
action.  In  this  way  there  arises  a  relative  lack  of  complement. 
This  can  occur  especially  if  part  of  the  amboceptors  has  become 
changed  into  an  amboceptoid  with  increased  affinity  (Wechsberg,1 
E.  Xeisser  and  Friedemann 2) .  In  bactericidal  experiments,  how- 
ever, the  cooperation  of  the  amboceptoids  has  not  yet  been  proved. 

The  completion  of  amboceptors  can  be  disturbed  in  another  way. 

1  Wiener  klin.  Wochensch.  1902.  2  Berl.  klin.  Wochensch.  1902. 


354  COLLECTED  STUDIES  IN  IMMUNITY. 

Thus  complement-diverting  groups  pre-existing  in  normal  serum  of 
the  species  in  question,  and  which  have  not,  therefore,  originated 
through  immunization,  may  be  present  or  may  be  set  free  by  the 
inactivation  (normal  anticomplements,  etc.).  The  question  which 
arises,  namely,  whether  one  is  dealing  with  a  deflecting  body  of  nor- 
mal serum  or  with  one  produced  by  immunization,  can,  of  course, 
be  decided  by  the  previous  investigation  of  the  normal  serum  of  the 
animal  in  question,  as  well  as  by  comparison  with  several  other  nor- 
mal sera  of  the  same  species 

In  all  of  these  cases,  however,  the  plates  with  the  largest  amounts 
of  immune  serum  will  show  the  least  bactericidal  action,  i.e.,  the 
largest  number  of  colonies.  From  this  it  follows  that  one  can  err 
in  judging  the  bactericidal  power  of  a  serum  if  only  larger  amounts 
of  immune  serum  are  used  for  the  bactericidal  test  (about  1.0,  0.3). 
Thus  in  the  beginning  we  overlooked  the  high  bactericidal  power 
of  a  dysentery  serum  (Shiga),  for  this  became  manifest  only  after 
we  employed  doses  of  0.025  immune  serum  and  still  less. 

The  deflection  of  complement  just  mentioned,  by  means  of  ambo- 
ceptors  produced  by  immunization  (or  by  amboceptoids),  permits 
of  another  method  of  testing  by  which  also  the  serum  can  be  shown 
to  be  a  specific  immune  serum.  For  this  purpose  one  uses  an  active 
normal  serum  bactericidal  in  itself  or  a  mixture  of  inactive  immune 
serum  and  a  complement.  By  means  of  a  preliminary  test  one 
determines  the  amount  of  serum  or  serum  mixture  which  completely 
kills  the  amount  of  culture  sown.  To  such  a  dose  of  serum  or 
serum  mixture  (bactericidal  in  itself)  decreasing  amounts  of  in- 
active immune  serum  are  added,  when  it  will  usually  be  found  that 
the  phenomenon  of  deflection  of  complement  again  appears.  This 
manifests  itself  by  the  fact  that  the  plates  with  the  larger  amounts 
of  immune  serum  show  a  larger  number  of  colonies,  the  number  of 
these  decreasing  in  proportion  with  the  amount  of  immune  serum 
added. 

In  order  to  interpret  the  results  of  the  plate  tests  correctly  it 
is  first  necessary  to  be  sure  whether  one  is  dealing  with  a  normally 
pre-existing  deflecting  body  or  with  one  produced  by  immunization 
(see  above).  By  means  of  combining  experiments  it  must  also 
be  shown  whether  the  deflection  is  caused  by  amboceptors  or  ambo- 
ceptoids. It  is  not  difficult,  by  binding  them  to  the  corresponding 
bacteria,  to  remove  the  amboceptors  produced  by  immunization. 
In  most  cases  the  addition  of  a  moderate  amount  of  bacteria  care- 


TECHNIQUE  OF  BACTERICIDAL  TEST-TUBE  EXPERIMENTS.  355 

fully  killed  (65°  for  J-l  hour)  and  centrifuged  will  suffice.  In  these 
cases,  however,  the  supernatant  fluid  must  always  be  examined 
microscopically  to  make  sure  that  all  the  bacteria  have  been  removed 
by  the  centrifuging.  For  any  such  dead  bacteria  loaded  with  ambo- 
ceptor, which  should  remain  in  the  fluid,  would  serve  to  deflect  com- 
plements in  the  further  course  of  the  experiment.  However,  in 
many  cases  it  is  possible  to  remove  all  the  bacteria  by  centrifuging. 
In  that  case  it  is  easy  to  show  that  the  bactericidal,  as  well  as  the  com- 
plement-deflecting power  of  the  serum,  has  disappeared  with  the  absorbed 
amboceptor.  If  only  the  deflecting  power  of  the  serum  remains, 
while  the  bactericidal  power  has  disappeared,  and  if  the  comparative 
test  has  shown  that  one  was  not  dealing  with  a  normal  anticom- 
plement  or  such  like,  we  conclude  that  a  complementophile  ambo- 
ceptoid  is  present,  one  which  has  originated  from  the  amboceptor 
produced  by  immunization. 

In  many  cases  in  which  a  plate  test,  as  it  has  previously  been 
decribed,  has  seemed  unsuited,  another  method  has  been  used  to 
overcome  the  difficulty.  Thus  after  allowing  the  immune  serum  to 
act,  instead  of  pouring  plates,  one  can  take  a  loop  from  each  test- 
tube  and  make  slant  agar  streaks.  If  one  the  nmerely  regards 
very  broad  results,  such  as  no  growth,  luxuriant  growth,  one  will 
obtain,  by  this  simple  means,  useful  comparative  values.  In  this 
way  Dr.  Lipstein  and  I  have  several  times  determined  the  power 
of  a  gonococcus  serum  which  we  produced  by  immunization. 


XXXI.  THE  PROPERTY  OF  THE  BRAIN  TO 
NEUTRALIZE  TETANUS  TOXIN.1 

By  Dr.  E.  MARX,  Member  of  the  Institute. 

WASSERMANN  and  Takaki's2  communication  stating  that  it  is 
possible  by  means  of  normal  brain  substance  to  decrease  the  toxicity 
of  tetanus  toxin,  or  even,  in  suitable  doses  to  entirely  neutralize  it, 
was  undoubtedly  of  great  theoretical  and  practical  significance. 
Their  statement  was  confirmed  by  many  different  investigators, 
Ransom,3  Metchnikoff,4  Marie,5  Blumenthal,6  Milchner,7  Danyz,8 
Zupnik,9  and  others.  These  experiments  were  devised  by  Wasser- 
mann  and  Takaki  as  a  test  for  the  correctness  of  the  side-chain  theory, 
according  to  which  the  cells,  susceptible  to  the  poison,  possess  recep- 
tors which  anchor  the  same.  They  argued,  if  the  theory  were  cor- 
rect, that  the  brain-cells  which  in  vivo  are  susceptible  to  the  poison 
should  also  be  capable,  at  least  in  the  fresh  state,  to  bind  the  poison 
in  vitro,  i.e.,  it  should  be  possible  to  neutralize  solutions  of  tetanus 
poison  with  brain  substance.  As  is  well  known  the  result  of  the 
experiments  agreed  with  the  theoretical  premises  and  they  were  so 
interpreted  by  Wassermann. 

This  interpretation  was  first  denied  by  Metchnikoff.  He  as  well 
as  Marie  had  repeated  Wassermann 's  experiments  and  conceded 


1  Reprint  from  the  Zeitsch.  f.  Hygiene  und  Infections-Krankheiten,  Vol.  40, 
1902. 

2  Berl.  klin.  Wochensch. 

3  Deutsch.  med.  Wochensch.  1898,  No.  5  (communicated  through  v.  Behring). 

4  Annales  de  1'Instit.  Pasteur,  1898,  pp.  81  and  263. 

5  Ibid,,  1898,  p.  91. 

6  Deutsch.  med.  Wochensch.  1898,  No.  12. 

7  Ibid.,  1898,  No.  16. 

8  Annales  de  PInstit.  Pasteur,  1899. 

9  Pra'ger  med.  Wochensch.  1899,  Nos.  14  and  15. 

356 


TETANUS  TOXIN  NEUTRALIZED  BY  BRAIN  SUBSTANCE.  357 

their  correctness,  but  on  the  basis  of  further  experiments  made  by 
Marie,  Metchnikoff  was  led  to  another  interpretation  of  the  results. 
Marie  found  that  when  poison  and  brain  substance  were  injected 
separately,  even  large  amounts  of  brain  substance  did  not  exert 
any  protection.  Metchnikoff,  therefore,  did  not  believe  in  any  neu- 
tralization of  poison  by  the  brain  substanc  in  vitro.  He  saw  the 
cause  of  the  apparent  neutralization  in  mixtures  of  tetanus  poison 
and  brain  substance  in  the  leucocyte-attracting  power  of  the  brain 
substance  injected  with  the  poison.  According  to  him  the  leuco- 
cytes were  the  agents  which  destroyed  the  poison,  and  the  brain 
substance  only  the  means  for  attracting  these. 

It  is  hardly  within  my  province  to  subject  these  experiments 
to  a  thorough  criticism;  that  must  be  left  to  those  directly  interested. 
I  should,  however,  like  to  mention  two  points  which  appear  to  me 
not  to  be  sufficiently  regarded.  First,  it  must  be  remembered  that 
with  a  dissolved  antitoxin  the  success  in  neutralization  on  mixing 
antitoxin  and  poison  in  vitro  is  considerably  higher  than  the  thera- 
peutic success  which  the  same  dose  attains  in  an  animal.  In  the 
above  experiments  there  is  added  to  this  the  fact  that  we  are  not 
dealing  with  a  dissolved  antitoxin.  On  the  contrary,  the  poison- 
neutralizing  power  is  exerted  by  a  mass  which,  from  experience,  we 
know  is  absorbed  with  great  difficulty. 

Subsequently  v.  Behring,  as  a  result  of  his  combining  experi- 
ments with  brain  substance,  expressed  doubts  as  to  the  correctness 
of  Wassermann's  explanation,  without,  however,  positively  taking 
either  one  side  or  the  other.  Basing  his  reasons  on  the  experiments 
of  Kitashima,  v.  Behring1  stated  his  views  as  follows: 

"If  an  emulsion  of  fresh  brain  substance  from  a  guinea-pig  is 
mixed  with  a  certain  dose  of  tetanus  poison,  a  dose  whose  power  is 
exactly  known,  it  will  be  found  that  with  small  amounts  the  poison 
will  completely  lose  its  poisonous  property;  with  larger  amounts 
there  is  a  distinct  decrease  of  this  property.  One  would  now  sup- 
pose that  large  amounts  of  poison,  whose  poisonous  property  has 
been  decreased  by  means  of  brain  emulsion,  would  require  less  anti- 
toxin for  their  neutralization  than  before  the  addition  of  the  brain 
emulsion.  But  this  is  by  no  means  always  the  case.  In  the  experi- 
ment— 

1  v.  Behring,  Allgemeine  Therapie  der  Infections-Krankheiten,  Part  I, 
p.  1033. 


358  COLLECTED  STUDIES  IN  IMMUNITY. 

0.008  cc.  poison  solution  No.  3, 
0.2  cc.  brain  emulsion; 

one  hour  later! 
Viooo  antitoxin  unit — 

we  not  only  found  no  excess  of  antitoxin,  but  found  that  the  injec- 
tion of  such  a  mixture  into  mice  caused  death  by  tetanus." 

The  result  of  this  experiment  led  v.  Behring  to  conclude  that 
further  study  of  the  poison-neutralizing  power  of  guinea-pig  brain 
would  probably  decide  the  question  in  favor  of  Metchnikoff's  views 
as  outlined  above.  A  subsequent  study  from  v.  Behring's  insti- 
tute demonstrated  that  a  union  evidently  takes  place  when  living 
brain  and  tetanus  poison  come  together. 

Ransom  1  studied  the  conditions  found  in  the  subarachnoid  space 
after  injections  of  tetanus  poison  or  tetanus  antitoxin.  It  would 
lead  us  too  far  to  recapitulate  these  brilliant  experiments,  and  I 
shall,  therefore,  content  myself  by  quoting  Ransom's  conclusions 
which  are  as  follows: 

"  These  experiments  strongly  corroborate  the  assumption  that 
tetanus  antitoxin  is  bound  in  the  central  nervous  system;  they  also 
indicate  that  this  union  takes  place  somewhat  gradually." 

There  is  surely  no  objection  to  our  placing  these  experiments 
on  the  living  brain  parallel  with  those  made  on  the  dead  brain.  It 
would  be  incomprehensible  for  a  brain,  removed  at  once  from  a 
freshly  killed  animal,  to  be  different  in  its  property  of  binding  tetanus 
poison  from  what  it  was  a  few  minutes  previously  in  the  living  animal. 

1  had    just  begun  a  study  in  this  institute  dealing  with   these 
problems,  but  discontinued   them  on   the  appearance  of  Ransom's 
paper  since  that  had  so  well  covered  the  subject. 

Some  time  after  this  Kitashima's  experiments  were  taken  up  by 
Gruber,2  although  without  re-examination.  In  these  experiments 
Gruber  saw  further  proof  of  the  incorrectness,  according  to  him,  of 
Ehrlich's  Side-chain  Theory.  In  response  to  this,  however,  Paltauf  3 
very  aptly  demonstrated  that  a  simple  calculation  will  show  that 
Kitashima's  experiments  cannot  in  any  way  be  regarded  as  con- 
clusive. He  expressed  himself  as  follows: 

J  Hoppe-Seyler's  Zeitschrift  fur  physiol.  Chemie  1900-1901,  Vol.  XXXI, 
p.  282  et  seq. 

2  Munch,  med.  Wochensch.  1901,  Nos.  46-49. 
8  Wiener  klin.  Wochensch.  1901,  No.  51. 


TETANUS  TOXIN  NEUTRALIZED  BY  BRAIN  SUBSTANCE.  359 

"0.008  cc.  tetanus  poison  No.  3+0.2  cc.  brain;  one  hour  later, 
Viooo  antitoxin  unit.  Tetanus  poison  No.  3  is  very  powerful. 
1  cc.  equals  5  million  mouse.  15  mouse  is  a  fatal  dose  for  a  mouse; 
in  the  experiment,  therefore,  40,000  mouse  or  more  than  2600Xthe  fatal 
dose  is  employed,  which  quantity,  to  be  sure,  is  neutralized  by  Viooo 
antitoxin  unit.  According  to  Wassermann,  however,  1  cc.  emulsion 
can  at  the  most  neutralize  10  fatal  doses;  according  to  others,  from 
30  to  100  fatal  doses.  Usually  1/5  cc.  suffices  to  neutralize  not  over 
20  doses  of  poison,  an  amount  which  is  very  minute  when  2600  doses 
of  poison  are  concerned." 

It  should  also  be  mentioned  that  Blumenthal  and  Wassermann l 
opposed  Gruber's  view.  Blumenthal  called  attention  to  the  fact 
that  when  brain  substance  is  added  to  a  toxin  solution  it  is  possible 
by  centrifuging  to  show  that  the  original  toxin  solution  has  been 
robbed  of  its  toxic  power,  a  result  which  cannot  be  obtained  with 
boiled  brain.  He  also  reminded  his  readers  that  he  had  shown  how, 
by  introducing  the  toxin  in  vivo,  the  power  of  the  brain  to  neutralize 
poison  had  been  diminished,  as  was  seen  on  testing  the  same  post- 
mortem. This  diminution  was  due  to  the  union  of  the  brain  sub- 
stance with  the  toxin,  and  was  in  proportion  to  the  amount  of  poison 
injected. 

Wassermann  too  is  still  convinced  that  there  is  a  chemical  union. 
His  view  is  also  borne  out  by  the  fact  that  in  the  rabbit,  in  which, 
according  to  the  researches  of  Donitz  and  Roux,  an  extensive  dis- 
tribution of  receptors  capable  of  binding  tetanus  toxin  was  to  be 
assumed,  other  organs  besides  the  brain  are  also  capable  of  neutraliz- 
ing the  poison  in  vitro.  This  is  in  direct  contrast  to  the  guinea-pig 
in  which  only  the  brain  possesses  this  power. 

In  view  of  all  this  we  determined  to  finally  decide  whether  on  the 
addition  of  brain  to  tetanus  poison  there  is  an  actual  union  of  poison, 
and  whether  if  this  is  so  there  is  a  summation  of  neutralizing  actions 
of  brain  and  antitoxin.  Our  old  studies  were  therefore  again  taken  up. 
We  began  with  the  re-examination  of  Kitashima's  experiments,  but 
under  such  conditions  that  the  errors  which,  independently  of  us, 
Paitauf  had  already  pointed  out,  namely,  the  employment  of  too 
large  doses  of  poison,  were  avoided. 

1  Deutsche  med,  Wochensch.  1902,  Vereinsbeilage,  No.  3. 


360  COLLECTED  STUDIES  IN  IMMUNITY. 


THE  MATERIAL  EMPLOYED,  AND  ITS  PREPARATION. 

In  these  experiments  a  great  deal  depends  on  the  manner  in  which 
the  brain  emulsion  is  prepared.  We  shall  therefore  again  describe 
the  method  in  detail,  although  Wassermann  and  Takaki  did  so  when 
they  reported  their  experiments. 

Each  guinea-pig  brain  was  thoroughly  mixed  with  10  cc.  0.85% 
salt  solution.  In  order  to  obtain  uniform  and  good  results  it  is  neces- 
sary that  the  emulsion  be  as  fine  as  possible.  For  this  purpose  the 
brain  substance  was  crushed  and  the  salt  solution  added,  at  first  drop 
by  drop,  until  a  fine  uniform  emulsion  resulted.  It  is  well  instead  of 
using  a  mortar  to  use  conical  glasses,  such  as  are  employed  at  the 
Rabies  Inoculation  Stations  for  preparing  the  fine  cord  emulsions  for 
injections.  These  conical  glasses  are  about  10  cm.  high  and  taper 
not  to  a  point,  but  to  a  hemispherical  surface  into  which  a  ground- 
glass  pestle  fits. 1  This  very  fine  emulsion  is  then  forced  through 
Herzberg  funnels,  such  as  are  used  in  testing  paper.  If  the  emulsion 
is  forced  through  the  finest  of  these,  fitted  with  wire-gauze  with  the 
smallest  mesh  obtainable,  it  will  be  found  that  the  emulsion  is  actually 
free  from  macroscopically  coarse  particles. 

The  poison  I  employed  was  a  tetanus  toxin  preserved  in  the 
institute  for  diagnostic  purposes.  This  poison,  I  may  add,  owing 
to  the  special  method  of  preparation,  differed  from  Behring's  test 
poisons  (at  least  from  those  which  can  be  obtained  in  the  market) 
in  being  free  from  spores.  This  fact  may  perhaps  not  be  without 
significance,  for,  under  the  conditions  which  here  obtain,  a  development 
of  the  spores  with  consequent  production  of  poison  in  the  animal  can- 
not be  denied  offhand. 

This  possibility  must  surely  often  be  counted  on.  It  was  for  this 
reason  that  Ehrlich  long  ago  allowed  only  such  tetanus  poisons  as 
were  freed  as  much  as  possible  from  spores  to  be  used  for  testing, 
and  for  exact  experimental  studies.  I  shall  soon  publish  an  account 
of  the  peculiarities  of  the  procedure  used  in  this  institute  for  obtaining 
such  poisons,  and  also  describe  a  method  for  preserving  tetanus  poison 
permanently,  which  we  have  found  very  useful. 

The  antitoxin  used  was  also  that  preserved  for  testing  purposes. 
1  grm.  contains  100  A.  E.  Behring. 

1  These  can  be  obtained  from  F.  and  M.  Lautenschlager,  Berlin,  N. 


TETANUS  TOXIN  NEUTRALIZED   BY  BRAIN   SUBSTANCE.  361 


METHOD  OF  MAKING  THE  EXPERIMENTS. 

The  method  employed  followed  exactly  in  principle  that  em- 
ployed by  Kitashima.  A  1  to  400  dilution  of  the  normal  solution 
of  the  poison  was  prepared.  To  each  cubic  centimeter  of  this,  which 
represents  forty  times  the  fatal  dose  for  a  mouse  of  15  grm.,  the 
desired  number  of  doses  of  brain  emulsion,  or  of  a  1 : 10  dilution  of 
this  emulsion  was  added,  the  fluid  made  up  to  2.5  cc.  by  the  addition 
of  0.85%  Nad  solution,  and  the  mixture  thoroughly  shaken.  At 
the  end  of  an  hour  0.5  cc.  of  the  dilutions  of  serum  in  question  were 
added  and  after  once  more  thoroughly  shaking,  ^-cc.  doses  of  this 
mixture  were  injected  subcutaneously  into  white  mice  weighing  15  grm. 
It  may  be  mentioned  that  in  the  controls  containing  only  brain  and 
poison  the  procedure  was  exactly  the  same  except  that  0.5  cc.  NaCl 
solution  were  added  at  the  end  of  the  hour  instead  of  0.5  cc.  serum. 
The  control  containing  only  poison  and  serum  was  treated  in  exactly 
the  same  manner  and  was  injected  in  the  usual  way  after  the  anti- 
toxin had  been  allowed  to  act  in  the  toxin  for  thirty  minutes.  It 
may  be  added  that  no  appreciable  difference  was  observed  if  the 
mixture  of  po  ison  +  bra  in + serum  was  injected  directly  after  the 
addition  of  the  serum  or  if  the  serum  was  allowed  to  act  on  the  brain 
+  poison  mixture  for  half  an  hour. 

RESULTS  OF  THE  EXPERIMENTS. 

My  results,  obtained  from  over  two  hundred  experiments  on 
mice,  do  not  furnish  the  slightest  ground  for  assuming  that  the  phe- 
nomenon found  by  Kitashima  is  the  rule.  On  the  contrary,  from 
my  experiments  I  can  positively  conclude  that  there  is  always  a 
summation  of  the  poison-neutralizing  action  of  the  brain  and  anti- 
toxin; furthermore  that  there  is  never  any  interference  with  the 
antitoxic  action  of  the  serum  as  a  result  of  the  previous  action  of 
the  brain  on  the  tetanus  poison.  This  fact  was  constantly  observed, 
no  matter  whether  large  or  very  small  doses  were  employed.  The 
series  of  tests  with  brain  emulsions,  as  well  as  those  with  brain  and 
poison  alone  without  serum,  do  not,  to  be  sure,  proceed  as  smoothly 
as  those  with  poison  +  serum;  however,  this  is  not  at  all  surprising; 
on  the  contrary,  it  is  quite  natural  that  the  particles  suspended  in 
the  emulsion,  even  if  they  are  very  fine,  cannot  produce  as  uniform 
effects  as  a  solution  of  antitoxin. 


362 


COLLECTED  STUDIES  IN  IMMUNITY. 


The  results  of  my  experiments  were  all  the  same  and  their  sig- 
nificance is  absolutely  clear.  From  the  large  number  of  tests  I  shall 
therefore  give  but  three.  These  will  incidently  show  the  well-known 
fact  that  the  power  to  neutralize  poison  is  often  very  different  in 
different  cases. 

TABLE  I. 


Degree  of  Dilution  of  the 
Serum. 

Control  Toxin  +  Serum. 

The  Experiment:  Toxin  +  1.5  cc. 
Brain  +  Serum. 

1:17500 
1:15000 
1:12500 
1:10000 
1:  8000 
1:  6000 
1:  4000 
Control:  only  toxin  and 
1.5  cc.  brain 

t3 

t4 
t4 

t9 
t9 
moderately  sick 

moderately  sick 
lightly  sick 

moderately  sick 

tt            it 

trace  sickness 

TABLE  II. 


Degree  of  Dilution  of  the 
Serum. 

Control  Toxin  +  Serum. 

The  Experiment:  Toxin  +  0.2  cc. 
Brain  +  Serum. 

1:17500 

t3 

moderately  sick 

1:15000 

t3 

ti             n 

:  12500 

t  4 

it             it 

:  10000 

t  4 

n             it 

:  8000 
:  6000 

very  severely  sick 
severely  sick 

ft             it 
trace  sickness 

:  4006 

moderately  sick 

1  1           it 

:  3000 

it             it 

well 

:  2000 

trace  sickness 

tt 

:   1000 

well 

11 

Control  :  only  toxin  and 

I 

, 

0.2  cc.  brain 

/ 

TABLE  III. 


Degree  of  Dilution  of  the 
Serum. 

Control 
Toxin  +  Serum. 

The  Experiment, 
Series  I.     Toxin  + 
0.1  cc.  Brain  + 
Serum. 

The  Experiment, 
Series  II.     Toxin  + 
0.2  cc.  Brain  + 
Serum. 

1:17500 
1:15000 
1:10000 
1:  5000 
Control:  only  toxin  + 
0.1  cc.  or  0.2  cc.  brain 

moderately  sick 

/             ~~ 

very  sick 

it       it 

it       it 
moderately  sick 

very  sick 

it       ii 

moderately  sick 

it            n 

TETANUS  TOXIX   NEUTRALIZED  BY  BRAIN   SUBSTANCE.  363 

All  of  these  experiments  show  that  the  mice  which  received  only 
toxin  and  brain  died,  whereas  additions  of  antitoxin  as  did  not  by 
themselves  suffice  to  neutralize  the  dose  of  poison  were  able  to  save 
the  animals  which  received  the  doses  of  brain  emulsion.  Hence 
the  action  of  the  brain  doses  (which  by  themselves  do  not  protect) 
adds  itself  to  that  of  non-protecting  doses  of  antitoxin  and  so  forms 
a  protective  dose. 

Resume. 

1.  The  neutralizing  effect  possessed  by  guinea-pig  brain  on  tetanus 
toxin  is  supplemented  by  that  of  antitoxin  when  these  are  allowed  to 
act  on  the  poison  in  vitro. 

2.  From  this  one  can  conclude  that  this  neutralizing  effect  of  guinea- 
pig  brain  on  tetanus  toxin  and  that  of  the  antitoxin  can  be  regarded 
as  equivalent  properties. 


XXXII.  THE    PROTECTIVE    SUBSTANCES    OF    THE 

BLOOD.1 

By  Professor  Dr.  P.  EHRLICH. 

MORE  than  ten  years  have  passed  since  the  studies  of  Fliigge 
and  of  Buchner  and  of  their  pupils  directed  attention  to  the  bac- 
tericidal substances  present  in  normal  blood  serum  and  their  rela- 
tion to  natural  immunity.  Buchner  especially  assumed  that  the 
serum  of  each  animal  species  contained  a  simple  definite  protective 
body,  the  alexin,  which  was  able  to  kill  off  foreign  cells,  especially 
bacteria  and  the  blood-cells  of  other  species;  that  this  acts  some- 
what after  the  manner  of  a  proteolytic  ferment  and  leaves  the  cell 
elements  of  its  own  species  unscathed.  The  recent  development 
of  the  doctrine  of  immunity,  inaugurated  by  v.  Behring's  discovery 
of  antitoxin,  has  also  shed  considerable  light  on  the  nature  of  pro- 
tective bodies  preformed  normally,  so  that  it  now  seems  advisable 
to  subject  the  mutual  relations  existing  between  these  to  a  closer 
analysis. 

There  can  hardly  be  any  doubt  that,  in  accordance  with  the 
principle  enunciated  by  Virchow  for  the  relation  existing  between 
cell  physiology  and  cell  pathology>  the  normal  protective  substances 
are  subject  to  the  same  developmental  laws  as  the  artificially  pro- 
duced antitoxic  and  bactericidal  substances.  It  is  obvious  that 
with  the  artificially  produced  protective  substances,  especially  with 
the  antitoxins,  it  will  be  far  easier  to  gain  an  insight  into  the  mechan- 
ism of  their  development,  for  in  this  case  one  possesses  not  only 
the  exciting  agent  (as,  for  example,  the  toxin),  but  also  the  resulting 
specific  product  (the  specific  antitoxin),  making  it  possible  to  study 
their  mutual  chemical  relations. 

1  Address  delivered  in  the  general  session  of  the  73d  Congress  of  German 
Naturalists  and  Physicians,  Hamburg,  Sept.  25,  1901.  (Reprinted  from  the 
Deutsche  med.  Wochenschrift  1901,  Nos.  51  and  52.) 

364 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  365 

This,  however,  is  not  possible  in  the  case  of  the  substances  natu- 
rally present,  and,  considering  the  complicated  chemistry  of  the 
living  organism,  we  shall  probably  long  continue  to  be  ignorant 
of  the  substances  which  act  as  the  physiological  excitants. 

Hence  it  is  not  a  mere  coincidence  that  the  attempt  to  formu- 
late a  theory  for  the  development  of  the  protective  substances  suc- 
ceeded first  in  connection  with  those  artificially  produced.  This  is 
now  well  known  as  the  side-chain  or  receptor  theory.  According 
to  my  view  this  theory  is  also  of  the  highest  significance  for  the  con- 
ception  of  the  nature  of  the  alexins.  I  shall,  however,  first  outline 
my  views  on  this  subject  as  they  are  applied  to  the  formation  of 
antitoxin,  as  this  is  comparatively  the  simplest  to  study. 

There  were,  as  you  all  know,  chiefly  two  views  concerning  the 
formation  of  antitoxin,  namely,  the  hypothetical  metamorphosis  of 
toxin  into  antitoxin,  and  the  secretion  theory,  which  approaches 
somewhat  the  side-chain  standpoint.  The  former  was  based  on 
the  observation  that  the  antitoxin  excited  by  a  certain  toxin  acts 
only  against  just  this  toxin  and  against  no  other.  This  specific 
action  is  such  a  conspicuous  phenomenon  that  it  was  at  first  believed 
that  the  intimate  relation  of  toxin  to  antitoxin  could  only  be  explained 
by  assuming  the  toxin  itself  to  be  the  mother  substance  of  the  anti- 
toxin. So  even  to  this  day,  Buchner  maintains  the  view  that  the 
antitoxins  and  related  substances  do  not  correspond  to  preformed 
or  even  wholly  newly  formed  constituents  of  the  organism,  but  that 
they  are  non-poisonous  transformation  products  of  the  substances 
introduced  for  purposes  of  immunization.  In  this  case,  therefore, 
the  relationship  of  antibody  to  the  substances  exciting  its  produc- 
tion would  be  due  to  a  similarity  of  the  two  components.  In  other 
words,  there  would  be  no  antagonism  such  as  exists  between  acid  and 
base,  but  an  attraction  of  like  to  like,  as  is  seen,  for  example,  in  poly- 
merization, in  the  attraction  of  crystallization,  or  in  the  structure 
of  starch  granules. 

Against  this  I  should  like  to  point  out  that  this  assumption  can- 
not apply  even  from  a  purely  chemical  standpoint  because  the 
processes  advanced  as  analogous  occur  in  concentrated  solutions, 
while  neutralization  of  toxin  and  antitoxin  takes  place  in  extremely 
dilute  solutions. 

The  biological  conditions,  however,  constitute  the  most  serious 
objection  to  the  assumption  of  a  transformation  of  toxin  into  anti- 
toxin. First  comes  the  enormous  difference  in  quantity  which  may 


366  COLLECTED  STUDIES  IN  IMMUNITY. 

exist  between  the  toxin  introduced  and  the  resulting  antitoxin. 
Knorr,  for  example,  has  shown  that  the  injection  of  tetanus  toxin  into 
a  horse  is  followed  by  the  production  of  an  amount  of  antitoxin  which 
would  neutralize  100,000  times  the  dose  ot  poison  employed.  Such 
an  enormous  disproportion  cannot  be  reconciled  with  Buchner's  view, 
according  to  which  each  part  of  toxin  would  make  an  antitoxin 
equivalent.  This  ratio  can  be  explained  only  by  a  theory  which 
makes  the  production  of  antibody  more  independent  of  the  exciting 
agent. 

Another  fact,  which  cannot  be  reconciled  with  a  transformation 
of  toxin  into  antitoxin,  is  the  marked  difference  existing  between 
so-called  active  and  passive  immunity.  If,  for  example,  by  injecting 
an  animal  with  poisons  or  bacteria  an  active  immunity  is  produced, 
this  immunity  may  in  favorable  cases  persist  for  years,  while 
in  passive  immunity  the  preformed  antitoxin  introduced  into  the 
organism  exists  but  a  short  time.  Such  a  difference  could  not  exist 
if  the  antitoxin  were  nothing  else  than  transformed  toxin;  for  in 
that  case  it  should  be  absolutely  immaterial  how  the  antitoxin  now 
in  the  organism  had  originated.  The  difference,  however,  depends 
on  the  fact  that  in  active  immunity  the  tissues  of  the  body  con- 
stantly produce  new  antitoxin,  keeping  pace  with  the  excretion  of 
the  same. 

This  production  of  the  antitoxin  by  the  body-cells  is  further- 
more confirmed  by  the  interesting  experiments  of  Roux  and  Vaillard, 
and  of  Salomonsen  and  Madsen.  They  took  an  animal  which  had 
been  actively  immunized,  and  whose  serum  showed  a  constant  amount 
of  antitoxin,  and  by  means  of  repeated  venesection  abstracted  a 
considerable  portion  of  its  blood.  In  case  the  antitoxin  had  been 
derived  from  the  toxin  introduced  there  should,  now  that  the  last 
traces  of  poison  had  disappeared  from  the  body,  have  been  a  marked 
loss  of  antitoxin  from  the  blood.  On  the  contrary  within  a  short 
time  it  was  found  that  the  amount  of  antitoxin  had  again  reached 
its  previous  level.  Another  point  in  support  of  the  assumption  that 
the  body-cells  produce  the  antitoxin  is  an  experiment  of  Salomonsen 
and  Madsen,  which  shows  that  the  amount  of  antitoxin  present  in  the 
blood  of  an  actively  immunized  animal  is  increased  if  the  animal  is 
treated  with  substances  which  increase  the  secretion  of  blood- cells 
in  general,  e.g.  pilocarpine.  This  experiment  was  advanced  by 
Salomonsen  and  Madsen  as  absolutely  opposed  to  the  transformation 
hypothesis  and  supporting  their  secretion  theory. 


THE   PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  367 

There  is  one  fact,  however,  by  which  the  transformation  hypothesis 
is  especially  refuted,  namely,  that  antitoxins  can  occur  in  the  blood 
of  normal  individuals.  Thus  diphtheria  antitoxin  is  found  in  the 
blood  of  horses  in  about  20-30%  of  the  animals  examined,  although 
diphtheria  infection  is  surely  a  rare  exception  with  these  animals. 
Horse  serum  furthermore  contains  antibodies  against  one  of  the  poisons 
produced  by  tetanus  bacilli,  tetanolysin,  but  not  against  the  tetanizing 
poison  of  the  same  bacilli,  the  tetanospasmin,  although  the  immune 
serum  artificially  produced  contains  both  antibodies. 

Just  these  observations,  which  can  easily  be  extended,  show  that 
even  the  normal  organism  can  produce  true  antitoxins  without  the 
intervention  of  the  corresponding  bacterial  substances.  Hence  these 
antibodies  cannot  be  transformation  products  of  the  poisons  injected, 
but  are  products  of  normal  cell  activity.  The  explanation  especially 
of  these  normal  processes  constitutes  one  of  the  chief  points  in  the  side- 
chain  theory. 

This  theory  is  based  primarily  on  a  thorough  analysis  of  the 
relations  between  toxin  and  antitoxin.  It  was  found,  by  means  of 
test-tube  experiments  with  ricin  and  related  bodies  which  act  on  red 
blood-cells,  that  it  was  extremely  probable  that  tOxin  and  antitoxin 
act  chemically  directly  on  each  other,  forming  a  new  innocuous  com- 
bination. It  was  now  necessary  to  study  the  neutralization  of  these 
two  substances  in  all  directions  in  great  detail.  For  this  purpose  I 
chose  diphtheria  toxin  and  antitoxin,  because  the  guinea-pig  organism, 
furnishes  such  a  uniform  test  object  for  this  poison  that  exact  quan- 
titative determinations,  such  as  are  used  in  physics  and  chemistry, 
are  attainable  in  animal  experiments.  The  limit  of  error  in  the  titra- 
tion  of  diphtheria  serum  titrations  is  not  more  than  1%,  surely  an 
astonishing  result  if  we  consider  that  we  arc  dealing  with  substances 
which  chemically  as  yet  are  entirely  unknown. 

The  results  which  I  obtained  in  the  earlier  years  of  my  investiga- 
tions were  really  very  discouraging,  for  they  seemed  to  present  an 
insurmountable  obstacle  for  the  chemical  conception.  In  chemical 
processes  when  two  substances  unite  to  form  a  third  substance, 
in  accordance  with  the  laws  of  stoichiometry,  we  must  insist  that  these 
components  act  on  one  another  in  definite  equivalent  proportions. 
In  the  action  of  diphtheria  antitoxin  or  toxin,  however,  this  law 
seemed  to  be  utterly  disregarded.  Thus  in  twelve  different  toxic 
bouillons  I  first  determined  the  quantity  which  was  neutralized  by  a 
constant  amount  of  antitoxin;  in  certain  instances  by  the  official 


368  COLLECTED  STUDIES  IN   IMMUNITY. 

standard  unit  of  antitoxin.  The  figures  thus  obtained,  as  was  to  be 
expected,  varied  greatly:  in  one  case  the  antitoxin  unit  neutralized 
0.25  cc.  toxic  bouillon,  in  another  case  1.5  cc.  This  is  not  in  the  least 
surprising,  for  it  is  well  known  that  the  amount  of  poison  given  off  by 
the  bacteria  to  the  medium  depends  on  the  strain  of  the  bacilli,  on 
the  preparation  of  the  bouillon,  etc.,  so  that  strong  poisons  and  weak 
poisons  arise.  But,  assuming  that  the  toxin  molecule  follows  chemical 
laws  in  its  union  with  antitoxin,  it  was  to  be  expected  that  in  the 
different  poisons  the  amounts  neutralized  by  1  I.  E.  (Immun  Einheit= 
Immune  Unit),  and  designated  as  LQ,  would  contain  equal  amounts 
of  true  poison,  or  in  other  words  that  the  various  poisons  which  differ 
in  their  L0  doses  represent  nothing  more  than  more  or  less  concentrated 
solutions  of  the  same  toxic  substance.  The  amount  of  poison  con- 
tained in  a  solution  is  measured  in  poison  units,  i.e.,  that  amount  of 
toxic  bouillon  which  just  suffices  to  kill  a  guinea-pig  weighing  250  grm. 
in  four  days.  Thus  if  in  a  certain  poison  A  we  find  the  amount  neu- 
tralized by  1  antitoxic  unit,  i.e.,  the  L0  dose,  to  be  1  cc.,  and\if  we 
further  find  that  0.01  cc,  of  the  same  poison  suffices  to  kill  a  guinea-pig, 
we  say  that  in  this  poison  the  L0  dose  represents  100  poison  units. 
In  accordance  with  the  law  of  equivalent  proportions  we  should  have 
expected  that  the  LQ  dose  of  the  various  poisons  would  contain  the 
same  number  of  poison  units.  As  a  matter  of  fact,  however,  the  result 
was  quite  the  reverse,  for  we  found  that  the  number  of  poison  units 
contained  in  L0  varied  from  a  minimum  of  10  units  to  a  maximum 
of  150.  According  to  the  view  held  at  that  time  that  the  antitoxin 
was  bound  only  by  the  toxin,  this  wide  divergence  from  the  laws  of 
equivalence  could  not  help  but  cause  the  assumption  that  the 
relations  existing  between  these  two  opposing  substances  were  other 
than  purely  chemical  ones. 

Finally  by  employing  a  method  of  study  which  has  proved  of 
considerable  value  in  scientific  investigations,  namely,  the  genetic 
method,  I  succeeded  in  getting  some  light  on  this  subject.  Follow- 
ing this  1  subjected  one  and  the  same  toxic  bouillon  to  comparative 
tests  at  different  times.  1  may  be  permitted  to  demonstrate  this  by 
means  of  a  simple  schematic  example.  In  a  freshly  made  poison 
we  find  that  the  quantity  which  is  neutralized  by  1  I.  E.,  in  other 
words  the  LQ  dose,  amounts  to  1  cc.,  and  that  this  contains  100  poison 
units.  If  the  same  poison  is  examined  at  the  end  of  about  six  months, 
it  is  found  that  the  L0  dose  is  the  same,  but  that  this  contains  only 
50,  i.e.,  half  the  number  of  toxic  doses.  That  is  to  say,  the  toxic 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  369 

bouillon  still  possesses  the  original  neutralizing  power  but  a  weaker 
toxic  action.  Hence  toxic  action  on  an  animal  arid  combining  power 
for  antitoxin  must  be  two  different  functions,  the  former  remaining 
constant  and  the  latter  decreasing. 

If  we  regard  these  conditions  from  the  chemical  standpoint,  we 
shall  see  that  they  are  most  readily  explained  by  assuming  that  the 
toxin  molecule  produced  by  the  diphtheria  bacilli  contains  two  dif- 
ferent groups,  of  which  one,  termed  the  haptophore  group,  effects 
the  union  with  antitoxin,  while  the  other,  the  toxophore  group, 
represents  the  actual  cause  of  the  toxicity.  These  two  groups  also 
differ  in  their  stability,  for  the  toxophore  group  is  very  unstable 
the  haptophore  group  far  more  stable.  Modified  poisons  in  which 
there  has  been  a  destruction  of  the  toxophore  group  while  the  hap- 
tophore group  has  been  preserved,  and  which  have  therefore  com- 
pletely lost  their  toxic  action,  are  called  "toxoids." 

The  presence  of  such  toxoids  fully  explains  the  apparent  devia- 
tions from  the  Jaws  of  equivalence  which  are  observed  in  neutralizing 
tests  with  toxin  and  antitoxin.  This  furnishes  new  and,  to  my  mind, 
incontrovertible  proof  for  the  chemical  view  of  the  process  of  neutral- 
ization. 

In  diphtheria  poison  at  least,  for  reasons  into  which  I  cannot 
here  enter,  it  seems  that  the  affinity  of  the  haptophore  group  of  the 
toxoid  molecule  for  the  antitoxin  is  exactly  the  same  as  that  of  the 
unchanged  toxin.  This  indicates  that  the  two  functionating  groups 
of  the  toxin  molecule  possess  a  certain  degree  of  independence.  I 
have  tried  further  by  means  of  refined  investigating  methods,  such 
as  partial  neutralizations,  to  extend  the  views  concerning  the  con- 
stitution of  the  poison  molecule.  My  observations,  so  far  as  the 
facts  are  concerned,  have  been  completely  confirmed  from  various 
sources.  Mention  should  be  made  especially  of  the  excellent  study 
ot  Madsen  on  diphtheria  toxin  and  tetanus  toxin,  and  of  the  inter- 
esting experiments  recently  published  by  Jacoby  on  ricin  and  its 
toxoids. 

In  studying  the  two  groups  of  the  poison  molecule,  we  are  con- 
cerned not  only  with  a  satisfactory  explanation  for  the  process  of 
neutralization.  The  presence  of  these  groups  gives  us  an  insight 
both  into  the  nature  of  the  poisoning  and  the  origin  of  the  antitoxin. 

So  far  as  this  last  point  is  concerned,  two  facts  in  particular 
indicate  that  the  haptophore  group  takes  a  leading  part  in  the 
immunity  reaction  in  the  organism,  viz  ,  (1)  the  observation  that 


370  COLLECTED  STUDIES  IN   IMMUNITY. 

toxoids,  which  lack  the  toxophore  group,  are  still  capable  of  exciting 
the  production  of  typical  antitoxins,  and  (2)  that  toxins  whose 
haptophore  group  is  preoccupied  by  antitoxins  lose,  as  a  result  of 
this  procedure,  their  power  to  produce  antitoxins.  Now  in  order  to 
understand  the  essential  role  played  by  the  haptophore  group  in  the 
formation  of  antitoxins  and  of  the  antibodies  in  general,  it  is  neces- 
sary above  all  to  study  the  other  side  of  this  question,  namely,  the 
functions  of  the  living  organism  in  the  formation  of  antibodies. 

The  demonstration  that  it  is  the  haptophore  group  of  the  toxin 
molecule  that  excites  the  production  of  immunity  leads  us  at  once 
to  regard  the  process  of  assimilation  of  the  living  cells  as  most  im- 
portant in  our  study.  Since  the  beginning  of  medicine  it  has  been, 
and  still  is,  generally  accepted  that  chemical' substances  can  act  only 
on  those  organs  with  which  they  are  capable  of  entering  into  closer 
chemical  relations.  In  his  "Cellular  Pathology/'  Virchow  expressed 
this  view  in  his  usual  clear  and  forcible  manner:  "Just  as  the  single 
cell  of  a  fungus  or  an  alga  abstracts  from  the  fluid  in  which  it  lives 
as  much  and  the  kind  of  material  as  it  needs  for  its  vital  processes, 
so  also  the  tissue  cell  within  a  compound  organism  possesses  elective 
properties  by  virtue  of  which  it  disregards  certain  substances  and 
takes  up  and  utilizes  others." 

"We  also  know  that  there  are  a  number  of  substances  which  have 
a  special  attraction  for  the  nervous  system  when  introduced  into  the 
body;  that  even  among  this  group  there  are  substances  which  pos- 
sess intimate  relations  to  certain  particular  parts  of  the  nervous 
system,  some  to  the  brain,  others  to  the  spinal  cord  or  to  the  sym- 
pathetic ganglia,  a  few  to  certain  special  parts  of  the  brain,  cord,  etc. 
1  may  mention  morphine,  atropine,  curare,  strychnine,  digitalin.  On 
the  other  hand  we  know  that  certain  substances  are  intimately 
related  to  certain  organs  of  secretion,  that  they  permeate  these 
secreting  organs  with  a  certain  selective  action,  that  they  are  ex- 
creted by  them,  and  that  when  supplied  in  excess  such  substances 
cause  an  irritation  in  these  organs/' 

It  is  remarkable  that  this  axiom  was  not  re-echoed  in  the  develop- 
ment of  scientific  pharmacology,  and  that  only  within  the  last  ten 
years,  thanks  to  the  labors  of  Hofmeister,  Overton,  Spiro,  Hans 
Meyer  and  myself,  an  improvement  has  taken  place  in  this  respect. 

According  to  these  newer  researches  there  is  not  the  least  doubt 
that  the  causes  of  this  elective  lodgment  in  certain  cell  domains  are 
not  all  of  the  same  nature.  In  general  the  modern  pharmacological 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  371 

school  now  believes  that  the  substances  ordinarily  foreign  to  the 
organism,  such  as  the  indifferent  narcotics,  alkaloids,  antipyretics, 
antiseptics,  do  not  effect  a  firm  chemical  union  with  the  body  ele- 
ments, but  that  their  distribution  follows  the  laws  of  solid  solutions 
or  of  the  formation  of  a  loose  salt.  In  the  case  of  the  poisons  acting 
on  the  central  nervous  system  it  is  especially  the  fat-like  substances 
of  the  nerve  tissue,  the  so-called  lipoids,  which  take  up  the  narcotics, 
just  as  ether  takes  up  the  alkaloids  in  the  Stas-Otto  procedure  of 
detecting  poisons.  There  are  a  number  of  reasons  in  support  of  the 
view  that  the  pharmacological  agents  in  question  are  stored  up  un- 
changed in  the  cells  or  in  certain  constituents  thereof,  especially  in 
those  similar  to  tat. 

Xaturally  this  does  not  deny  the  possibility  that  certain  sub- 
stances foreign  to  the  body  may  enter  an  albumin  molecule  by  sub- 
stitution. Thus  if  protoplasma  is  treated  with  nitric  acid  the  nitro 
group  enters  the  albumin  radicle,  giving  rise  to  a  yellow  color.  Such 
substitutions,  however,  in  the  conditions  under  which  pharmacolog- 
ical actions  can  occur,  will  usually  only  be  effected  by  combinations 
possessing  high  internal  tension  and  for  that  reason  capable  of  such 
addition  reactions.  This  may  perhaps  be  the  case  with  vinylamin^ 
which,  according  to  Levaditi's  experiments  conducted  in  my  labora- 
tory, produces  necrosis  of  the  renal  papillae  in  a  large  number  of 
animals,  a  phenomenon  probably  to  be  ascribed  to  such  a  chemical 
anchoring. 

The  ordinary  medicinal  substances,  however,  are  not  so  constructed 
that  they  can  produce  such  energetic  sections.  In  general  we  may 
assume  that  chemo-synthetic  processes  do  not  play  a  prominent  part 
in  their  distribution. 

It  may,  however,  be  regarded  as  an  absolute  fact  that  synthetic 
processes  play  an  important  role  in  the  life  of  the  cell  in  another 
direction.  If  by  boiling  certain  cell  material  with  acids  we  are  able 
to  split  off  certain  definite  groups  (such  as  those  of  sugar,  etc.), 
this  fact  proves  the  chemical  character  of  this  combination.  As  a 
matter  of  fact  the  two  series  of  phenomena  which  we  are  here  dealing 
with  have  long  been  separated  by  general  custom.  The  term  assim- 
ilability  is  reserved  exclusively  for  those  substances  which  are  an- 
chored by  the  cells  synthetically,  and  which  in  this  way  become  con- 
stituents of  the  protoplasm.  No  one  would  think  of  speaking  of 
morphine,  or  of  methylene  blue,  substances  which  enter  into  certain 
cells  and  lodge  there,  as  being  assimilable. 


372  COLLECTED  STUDIES  IN   IMMUNITY. 

These  explanations  will  suffice  to  show  that  the  term  assimila- 
bility,  as  I  employ  it,  is  restricted  somewhat  more  than  is  customary, 
for  I  reserve  it  exclusively  for  the  specific  nutritive  substances  of  the 
living  protoplasm.  According  to  this  view  the  process  of  cell  assimila- 
tions is  a  synthetic  one  which  presupposes  the  presence  of  two  groups 
effecting  the  synthesis  and  having  a  strong  chemical  affinity  for  each 
other. 

Hence  I  assume  that  the  living  protoplasm  possesses  side-chains 
or  receptors  which  possess  a  maximum  chemical  affinity  for  certain 
particular  groups  of  the  specific  nutritive  substances,  and  that  they 
therefore  anchor  these  substances  to  the  cell.  The  receptor  apparatus 
of  the  cells  is  highly  complicated,  the  red  blood-cell,  for  example, 
possessing  perhaps  a  hundred  different  types  of  receptors. 

If  this  view  is  accepted  and  it  is  recalled  that  in  the  toxin  mole- 
cule it  is  the  haptophore  group  which  effects  the  development  of 
immunity,  only  a  very  small  step  is  required  in  order  to  gain  an 
insight  into  the  nature  of  antitoxin  formation.  This  is  the  very 
natural  assumption  that  among  the  various  receptors — perhaps  by 
chance — the  haptophore  group  of  the  toxin  finds  one  which  possesses 
an  especial  affinity  for  this  haptophore  group.  It  is  not  at  all  neces- 
sary that  every  bacterial  toxin  should  find  fitting,  i.e.  toxophile, 
receptors  in  every  animal  species.  On  the  contrary  just  this  absence 
of  receptors  constitutes  one  of  the  reasons  why  certain  animal  species 
are  immune  against  certain  particular  poisons.  Furthermore,  all 
the  facts  indicate  that  the  susceptibility,  i.e.  the  receptiveness,  of  an 
organism  for  a  certain  toxin  is  associated  with  the  presence  of  such 
toxophile  groups  of  the  protoplasm,  a  point  which  finds  suitable 
expression  in  the  term  receptors. 

As  a  result  of  anchoring  the  toxin  molecule  by  .neans  of  the 
haptophore  group  the  cell  is  influenced  in  two  directions.  Primarily 
owing  to  the  lasting  influence  of  the  toxophore  group,  it  sickens,  a 
condition  which  manifests  itself  by  disturbed  functions  and  possibly 
by  pathological  anatomical  changes.  Besides  this,  however,  in  a 
manner  shortly  to  be  discussed,  a  regenerative  process  is  begun  which 
can  lead  to  the  formation  of  antitoxin.  Since  this  regenerative 
process  can  be  excited  by  toxoids  lacking  the  toxophore  group,  as 
well  as  by  the  toxins  themselves,  we  must  assume  that  it  is  inti- 
mately related  to  the  haptophore  group.  Hence  the  two  parallel 
processes,  antitoxin  production  and  toxic  action,  are  independent  in 
that  they  are  caused  by  two  different  groups.  In  harmony  with  this 


THE   PROTECTIVE  SUBSTANCES  OF  THE  BLOOD  373 

is  the  fact  that  the  two  processes  may  interfere  with  one  another; 
a  marked  pathological  action  can  diminish  the  regenerative  process 
or  even  prevent  it  entirely.  This  is  shown,  for  example,  by  the  fact 
that  it  is  almost  impossible  in  the  case  of  certain  animals  highly 
susceptible  to  tetanus  poison,  such  as  mice  and  guinea-pigs,  to  pro- 
duce antitoxin  by  means  of  unmodified  poison,  while  the  result  is 
easily  attained  by  the  use  of  toxoids. 

Coming  now  to  the  regenerative  process,  which  leads  to  the  pro- 
duction of  antitoxin,  it  will  be  found  by  any  one  familiar  with  the 
fundamental  principles  formulated  by  Carl  Weigert  that  there  is 
nothing  remarkable  about  the  process.  The  receptor  which  has 
anchored  the  haptophore  group  of  the  toxin  or  toxoid  molecule 
becomes  useless  for  the  cell  because  of  this  occupation;  it  is  no  longer 
able  to  exercise  its  normal  function,  namely,  the  anchoring  of  nutri- 
tive substances.  The  cell  has  thus  suffered  a  loss  which  must  be 
replaced. 

In  such  processes  it  is  very  common  to  find,  as  Weigert 's  re- 
searches have  shown,  that  the  loss  is  not  merely  replaced,  but  that 
it  is  over  compensated.  The  same  thing  takes  place  in  the  methodical 
immunization  when  continued  and  ever  increased  doses  of  immu- 
nizing substance  are  introduced.  Part  of  the  newly  formed  re- 
ceptors still  attached  to  the  cell  are  occupied  by  the  immunizing 
substance  only  to  be  replaced  by  a  regeneration  greater  in  degree 
than  before.  Owing  to  this  increased  demand  the  protoplasm  to  a 
certain  extent  is  trained  in  one  direction,  namely,  to  produce  anew 
a  certain  kind  of  constituent,  the  receptors  in  question.  Finally, 
such  an  excess  of  receptors  is  produced  that  there  is  no  longer  room 
in  the  protoplasm  for  them.  Then  they  are  thrust  off  as  free  mole- 
cules and  pass  into  the  body  fluids.  According  to  this  view  the  anti- 
toxin is  nothing  more  than  the  thrust-off  receptor  apparatus  of  the 
protoplasm,  i.e.,  a  normal  cell  constituent  produced  in  excess. 

From  among  the  many  facts  already  at  hand  I  shall  select  merely 
a  few  to  serve  as  proof  of  the  correctness  of  this  hypothesis,  this 
"side-chain  theory,"  as  it  is  called. 

The  first  point  deals  with  the  demonstration  in  normal  tissues 
of  the  toxinophile  receptors  assumed  by  the  theory.  Although  such 
an  anchoring  of  the  poison  by  the  organs  had  already  been  demon- 
strated by  the  clinical  course  of  the  poisoning  and  by  Donitz's  thera- 
peutic experiments  on  animals  poisoned  with  tetanus  and  diphtheria 
poisons,  it  remained  for  Wassermann  to  show  that  certain  body 


374  COLLECTED  STUDIES  JN   IMMUNITY. 

elements  anchor  the  toxin  even  in  a  test-tube  and  neutralize  the 
toxin  just  as  does  the  antitoxin.  If  he  added  crushed  fresh  guinea- 
pig  brain  to  tetanus  toxin,  he  found  that  the  brain  substance  anchored 
the  toxin  in  such  a  manner  that  not  only  was  the  supernatant  fluid 
robbed  of  its  toxic  action,  but  that  the  brain  laden  with  tetanus 
toxin  also  exerted  no  toxic  effect.  From  this  we  can  conclude  that 
a  chemical  union  has  taken  place  between  constituents  of  the  ganglion 
cells  and  the  tetanus  toxin.  This  combination  is  so  firm  that  it  is 
not  broken  up  on  being  introduced  into  the  animal  body;  as  a  result 
the  toxin  remains  innocuous. 

That  this  is  really  a  specific  reaction  and  not,  for  instance,  merely 
an  absorption  is  shown  by  the  fact  that  boiled  brain,  in  which  the 
chemical  groups  in  question  are  destroyed,  is  just  as  little  able  to 
exert  this  action  as  the  pulp  of  any  other  organ  of  the  guinea-pig. 

In  addition  to  this  Ransom  has  shown  that  the  brain  of  living 
animals  possesses  the  same  toxin-destroying  power.  In  view  of 
this  it  would  appear  that  the  objections  made  by  Danysz,  which 
refer  to  the  divergent  behavior  of  the  decomposed  brain  pulp,  possess 
no  great  significance.  I  will  not  deny  the  fact  that  the  favorable 
result  achieved  in  tetanus  is  evidently  due  only  to  the  coincidence 
that  the  tetanophile  receptors  are  present  in  large  quantity  in  the 
brain.  Such  a  coincidence,  of  course,  need  not  obtain  for  every 
poison.  If  the  organs  endangered  by  the  toxin  contain  only  small 
quantities  of  toxin  receptors  it  will  be  found  that  with  what  are, 
at  best,  very  coarse  experimental  methods  these  receptors  escape 
detection.  This  is  the  case,  for  example,  with  botulism  toxin  and 
diphtheria  toxin. 

Such  confusing  chance  occurrences  can,  however,  be  avoided 
with  certainty  if  one  employs  poisons  artificially  produced,  poisons 
which,  owing  to  their  mode  of  production,  are  directed  against  cer- 
tain particular  kinds  of  cells.  The  ha?molysins  produced  by  injec- 
tions of  blood,  spermotoxins,  and  numerous  other  cytotoxins  may 
serve  as  examples.  In  all  of  these  cases  it  can  positively  be  proved 
that  the  toxin  is  anchored  by  the  susceptible  cells  in  specific  fashion. 

The  second  point  concerns  that  premise  of  my  theory  which 
states  that  the  same  organs  which  possess  a  specific  affinity  for  the 
poison  molecule  are  able  to  produce  antitoxin.  In  this  connection 
the  very  neat  experiments  made  by  Romer  on  abrin  immunization 
should  be  mentioned.  As  is  well  known,  abrin,  the  toxalbumin  of 
jequirity  beans,  is  able  to  excite  marked  inflammation  of  the  con- 


THE   PROTECTIVE    SUBSTANCES  OF  THE  BLOOD.  375 

junctiva  in  man  and  animals.  I  have  shown,  furthermore,  that  it 
is  possible,  by  means  of  conjunctival  instillations,  to  actively  immu- 
nize rabbits  against  abrin.  Romer  immunized  a  rabbit  by  means 
of  rapidly  increased  doses  into  the  right  eye  and  killed  the  animal 
at  the  end  of  three  weeks.  It  was  then  found  that  the  conjunctiva 
of  the  right  eye  which  had  been  the  site  of  the  inflammatory  process 
was  able,  when  ground  up  with  a  suitable  amount  of  abrin,  almost 
completely  to  neutralize  the  action  of  this  poison,  whereas  the  other 
conjunctiva,  when  similarly  ground  up  with  abrin,  was  unable  to 
protect  the  animal  from  death.  From  this  Romer  rightly  concludes 
that  in  this  conjunctival  immunization  part  of  the  antitoxin  is  fur- 
nished by  the  conjunctiva  which  reacts  locally.  Aside  from  its 
theoretical  interest  I  believe  that  this  demonstration  of  the  local 
origin  of  antitoxin  at  the  site  of  injection  possesses  great  practical 
significance.  In  certain  cases  the  possibility  is  thus  given  to  trans- 
fer part  of  the  antitoxin  production  from  the  vital  organs  to  the  in- 
different connective  tissues.  7  . 

The  third  point  concerns  the  thrusting-off  of  the  surplus  receptors. 
A  prerequisite  for  this  thrusting-off  is  that  the  receptors  in  question, 
which  are  normally  firmly  attached  to  the  protoplasmal  molecule, 
become  loosened.  In  several  favorable  cases  it  has  been  possible 
to  confirm  this  postulate  of  my  theory  experimentally,  though  to 
be  sure  these  deal  with  immunization  by  bacteria  and  not  with  solu- 
ble poisons.  Pfeiffer  and  Marx  succeeded  in  showing  that  with  a 
suitably  conducted  cholera  immunization  it  is  possible  to  find  a 
period  at  which  the  blood  is  still  free  from  protective  substances, 
although  the  specific  protective  substances  can  be  abstracted  from 
the  blood-forming  organs  by  crushing  them  up  with  salt  solution. 
In  my  opinion  this  can  be  due  only  to  an  extraction  of  receptors 
which,  since  it  is  just  previous  to  their  extrusion,  are  only  loosely 
attached  to  the  protoplasmal  molecule. 

Almost  simultaneously  with  Pfeiffer  and  Marx,  the  same  results 
were  obtained  by  Wassermann  with  typhoid,  and  these  were  later 
confirmed  by  Deutsch.  In  all  of  these  experiments  the  hsemato- 
poetic  system  represents  the  site  of  production  of  these  antibodies. 
The  significance  of  this  circumstance  for  the  immunizing  process 
has  been  pointed  out  by  Metchnikoff's  teachings. 

These  few  examples  will  suffice  to  show  that  the  side-chain  theory 
has  fully  stood  the  test  of  experiment.  During  the  many  years  of 
my  experimental  activity  I  have  not  met  a  single  fact  which  con- 


376  COLLECTED  STUDIES  IN   IMMUNITY. 

tradicts  this  theory  and  might  serve  to  refute  it.  I  may,  there- 
fore, regard  the  theory  as  well  established  and  proceed  to  discuss  in 
detail  several  important  points  which  follow  from  it. 

The  side-chain  theory  explains  in  the  most  natural  fashion  the 
specific  relations  existing  between  toxin  and  the  corresponding  anti- 
toxin. Furthermore  the  theory  makes  the  immunizing  action  of  the 
antitoxins  perfectly  comprehensible.  When  injected  subcutaneously 
into  animals  in  the  usual  manner  the  poisons  are  brought  to  the 
organs  possessing  toxinophile  receptors  (susceptible  organs)  by 
means  of  the  circulation.  If,  however,  these  poisons  meet  with 
free  toxinophile  groups  in  the  blood,  they  will  at  once  combine  with 
the  same  and  so  be  diverted  from  the  susceptible  organs,  v.  Beh- 
ring  has  expressed  this  hypothesis  as  follows:  "The  same  substance 
which  when  in  the  cells  is  a  prerequisite  and  cause  of  the  poisoning 
becomes  the  healing  agent  when  present  in  the  blood." 

To  my  mind  we  are  here  dealing  with  a  general  biological  law 
which  is  not  limited  to  the  toxins  but  applies  to  a  great  many,  if 
not  to  all,  poisonous  substances.  I  need  only  cite  the  saponin  poison- 
ing of  red  blood-cells.  Ransom  found  that  the  blood-cells  take 
up  saponin  owing  to  their  content  of  cholesterin  and  are,  as  a  result, 
subjected  to  the  deleterious  action  of  the  poison,  whereas  certain 
sera,  which  exert  a  protection  against  saponin  poisoning  owe  this 
protective  property  to  the  same  cause,  namely,  the  presence  of  choles- 
terin in  the  serum. 

Furthermore  the  theory  at  once  explains  the  fact  that  the  tissues 
of  an  immunized  animal  are  subject  to  the  action  of  the  poison  when 
in  some  way  the  action  of  the  antitoxin  contained  in  the  serum  is 
prevented.  Thus  Roux  showed  that  rabbits  immunized  against 
tetanus  become  poisoned  just  as  rapidly  as  control  animals  if  the 
tetanus  poison  is  brought  into  direct  contact  with  the  brain-cells  by 
means  of  intracerebral  injections.  This  fact  is  demanded  by  my 
theory,  for,  just  as  in  immunized  animals,  the  ganglion  cells  contain 
an  excess  of  toxinophile  groups  and  are  thus  especially  adapted  to 
anchor  the  poison  which  injures  them.  It  was  a  grave  error  on  the 
part  of  Roux  to  suppose  that  this  experiment  controverted  the  side- 
chain  theory.  Roux  thought  that  according  to  my  view  a  consider- 
able amount  of  antitoxin  had  accumulated  in  the  brain-cells  and 
that  therefore  the  immunized  animals  should  possess  a  local  brain 
immunity.  There  is  evidently  a  misconception  as  to  the  term  ''anti- 
toxin." Just  as  we  cannot  term  any  mass  of  iron  a  lightning-rod, 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  377 

but  restrict  this  term  to  such  masses  of  iron  which  deflect  the  light- 
ning from  a  particular  point,  so  we  must  restrict  the  term  antitoxin 
to  those  toxinophile  groups  which  circulate  in  the  blood  and  thus 
deflect  the  poison  from  the  susceptible  organs.  The  toxinophile 
groups  present  in  these  susceptible  organs  are  not  toxin  deflectors  but 
toxin  attr actors. 

The  theory  also  explains  why  the  property  of  producing  antitoxins 
is  restricted  to  certain  products  of  metabolism  of  living  cells.  All 
experiments  to  produce  antibodies  by  means  of  chemically  well  de- 
fined toxic  substances,  such  as  morphine,  strychnine,  saponin,  etc., 
have  failed. 

If  we  bear  in  mind  that  the  distribution  of  these  substances  in 
the  organism  takes  place  without  chemical  union  and  therefore  with- 
out the  intervention  of  receptors,  the  negative  result  of  these  experi- 
ments will  not  surprise  us.  The  property  of  forming  antitoxin  is 
possessed  only  by  such  substances  as  possess  a  group  able  to  unite 
with  the  side-chains  or  receptors  which  effect  assimilation.  It  must 
be  remembered  that  all  the  poisons  which  excite  the  production  of 
antitoxin  are  highly  complex  products  of  animal  and  vegetable  cells, 
which  in  their  chemical  properties  approach  the  true  albumins  and 
peptones.  In  1897,  by  means  of  my  theory,  the  production  of  anti- 
toxin and  the  binding  of  foodstuff  were  first  brought  into  connec- 
tion. At  that  time  nothing  was  known  of  the  fact  that  even  ordinary 
foodstuffs  are  capable  of  an  analogous  action.  1  have  therefore  been 
able  to  regard  as  an  agreeable  confirmation  of  my  views  the  circum- 
stance that  this  consequence  of  my  hypothesis  has  actually  repeatedly 
been  demonstrated  within  the  past  year,  especially  by  Bordet. 

If  animals  are  injected  with  milk,  it  is  found  that  their  serum 
gains  the  property  of  precipitating  the  milk  in  curds.  This  precipita- 
tion is  also  strictly  specific,  since  numerous  experiments  show  that 
the  coagulating  serum  obtained  by  treatment  with  goat  milk  coag- 
ulates only  goat  milk,  and  not  the  milk  of  other  species,  as,  for  ex- 
ample, that  of  women  or  cows. 

The  results  are  similar  if  animals  are  injected  with  other  albumi- 
nous substances,  e.g.,  with  the  sera  of  different  species  or  with  egg 
albumin.     In  this  case  in  the  serum  of  the  animal  there  develop  sub 
stances  (termed  coagulins  or  precipitins)  which  specifically  precipitate 
the  corresponding  kind  of  albumin. 

Deviations  from  the  law  of  specificity  occur  only  in  so  far  as  the  sera  of 
closely  related  animal  species  contain  substances  more  or  less  similar  Thus 


378  COLLECTED   STUDIES  IN   IMMUNITY. 

the  coagulin  obtained  by  testing  rabbits  with  human  serum  precipitates  only 
human  serum  and  the  serum  of  the  nearest  related  species,  apes.  This  reaction, 
which  was  developed  especially  by  the  researches  of  Uhlenhuth  and  of  Wasser- 
mann,  was  therefore  proposed  tor  the  forensic  identification  of  blood. 

From  this  we  see  that,  entirely  in  harmony  with  my  views,  the 
injection  of  foodstuffs  is  followed  by  the  production  of  typical  anti- 
bodies, which  combine  with  the  exciting  agent  in  a  specific  manner. 
An  analogous  reaction  takes  place  in  the  normal  processes  of  cell 
nutrition  and  serves  as  the  chief  source  of  the  protective  substances 
present  in  normal  blood  in  such  great  numbers. 

The  conditions  become  much  more  complicated  than  those  just 
•described  if,  instead  of  the  relatively  simple  soluble  metabolic  prod- 
ucts, living  cell  material  is  employed.  This  is  the  case,  for  instance, 
in  immunization  against  cholera,  typhoid,  anthrax,  erysipelas  of  swine, 
.and  many  other  infectious  diseases. 

In  these  diseases  under  certain  circumstances  there  develop  many 
other  reactive  products  beside  the  antitoxins  produced  against  the 
bacterial  toxins.  The  reason  for  this  is  that  every  bacterium  is  a 
highly  complex  living  cell  which,  when  it  disintegrates  in  the  animal 
body,  gives  rise  to  a  large  number  of  different  components.  Of  these 
a  great  many  are  able  to  produce  antibodies. 

Hence  as  a  result  of  the  introduction  of  bacterial  cultures,  in 
addition  to  the  specific  bacteriolysins,  which  cause  a  solution  of  the 
bacteria,  we  see  substances  develop,  such  as  the  antiferments 
(v.  Dungern,  Morgenroth,  Briot),  the  much  discussed  agglutimns 
(Gruber,  Durham,  Pfeiffer),  and  the  coagulins  (Kraus,  Bordet), 
which  specifically  precipitate  certain  albuminous  substances  that 
have  passed  into  the  culture  fluid. 

The  most  interesting  and  important  of  the  substances  arising  in 
.such  an  immunization  are  undoubtedly  the  bacteriolysins,  which 
have  been  studied  especially  by  Pfeiffer  and  Bordet.  At  first  it  is 
highly  surprising  that  the  injection  of  cholera  vibrios  into  the  animal 
body  should  be  followed  by  the  formation  of  a  substance  which  is 
able  to  dissolve  the  cholera  vibrio,  and  only  thit>  bacterium.  This 
action  is  so  perfectly  adapted  to  the  purpose  and  is  apparently  .so 
novel  that  it  seems  to  fall  beyond  the  pale  of  the  normal  functions 
of  the  body.  It  was  therefore  of  the  highest  importance  to  explain, 
irom  the  standpoint  of  cellular  physiology,  the  origin  of  these  sub- 
stances also.  The  solution  of  this  problem  offered  considerable  diffi- 


THE   PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  379 

culties  and  did  not  succeed  until  the  hsemolysins  were  used  in  the 
experiments  in  place  of  the  bacteriolysins. 

Haemolysins  are  peculiar  poisons  which  destroy  red  blood-cells. 
Such  haemolysins  are  found  in  part  in  certain  normal  species  of  serum, 
in  part  they  can  be  produced  artificially,  as  will  be  subsequently 
described.  In  their  fundamental  properties  they  correspond  entirely 
to  the  bacteriolysins,  but  possess  the  great  advantage  over  the  latter 
in  that  they  readily  permit  the  employment  of  test-tube  experiments 
whereby  the  individual  variability  of  the  animal  body  is  excluded, 
and  so  allow  accurate  quantitative  determinations. 

Belfanti  and  Carbone  discovered  the  curious  phenomenon  that 
the  serum  of  horses,  after  they  had  been  treated  with  blood-cells  of 
rabbits,  contains  substances  which  are  highly  toxic  to  rabbits,  and 
only  to  these  animals.  Bordet  showed  that  the  cause  of  this  toxicity 
is  a  specific  haemolysin  directed  againt  the  rabbit  blood-cells.  He 
showed  further  that  such  haemolysins,  derived  by  injection  of  foreign 
blood-cells,  lose  their  power  to  dissolve  blood  when  heated  for  half 
an  hour  to  55°  C.  Bordet  found  also  that  the  hsemolytic  property  of 
such  inactivated  sera  is  again  restored  if  certain  normal  sera  are 
added.  These  important  observations  showed  a  complete  analogy 
between  these  phenomena  and  those  observed  with  bacteriolysins  by 
Pfeiffer,  Metchnikoff ,  and  especially  by  Bordet.  In  the  case  of  bacteri- 
olysins it  was  found  that  serum  freshly  drawn  from  a  goat  immunized 
against  cholera  is  able  to  effect  solution  of  cholera  vibrios,  i.e.,  to  give 
the  so-called  Pfeiffer  reaction.  Apparently  this  property  disappears 
spontaneously  if  the  serum  is  allowed  to  stand;  it  disappears  rapidly 
when  the  serum  is  heated  to  55°  C.  The  cholera  serum  rendered 
inert  by  heating  exerts  its  protective  power  in  the  animal  body  un- 
changed; and  in  test-tube  experiments  it  attains  its  original  solvent 
power  on  the  addition  of  small  amounts  of  normal  goat  or  guinea- 
pig  serum,  although  the  latter  do  not  by  themselves  injure  cholera 
vibrios. 

These  experiments  show  that  in  bacteriolysis  two  substances  act 
together;  one,  contained  in  immune  blood,  is  relatively  stable  and 
represents  the  carrier  of  the  specific  protective  action;  the  other,  pres- 
ent in  every  normal  serum,  is  easily  destroyed.  For  the  present 
the  'former  is  called  the  "immune  body,"  while  the  latter,  since 
it  complements  the  action  of  the  immune  body,  is  called  the  "com 
piemen  t." 

Since  the  hsemolysins  are  by  far  the  most  convenient  for  experi 


380  COLLECTED  STUDIES  IX   IMMUNITY. 

mental  study,  Dr.  Morgenroth  and  I  have  endeavored  in  these  to  dis- 
cover the  mode  of  action  of  these  two  components  on  the  susceptible 
object,  the  red  blood-cells.  For  this  purpose  we  first  prepared  solu- 
tions containing  either  only  the  immune  body,  or  only  the  complement. 
These  solutions  were  then  brought  into  contact  with  the  appropriate 
blood-cells,  after  which  the  fluid  and  blood-cells  were  separated  by 
means  of  the  centrifuge.  The  two  portions  were  then  tested  to 
determine  whether  these  substances  had  been  taken  up  by  the  blood- 
cells.  These  experiments  showed  that  the  blood-cells  are  incapable 
of  taking  up  complement  alone,  whereas  they  eagerly  take  up  the 
immune  body.  If,  however,  the  serum  contains  both  components 
they  are  both  bound  by  the  blood-cells  in  question. 

A  confirmation  of  this  fact  was  furnished  by  Bordet,  who  showed 
that  blood-cells  or  bacteria  which  by  previous  treatment  have  become 
loaded  with  immune  body,  abstract  the  complement  from  fluids  con- 
taining the  same  with  great  avidity.  These  facts  have  been  confirmed 
from  all  sides.  They  show  that  the  blood-cells,  or  the  bacteria,  anchor 
the  immune  body  but  not  the  complement,  but  that  the  complement 
is  also  bound  as  soon  as  the  immune  body  has  been  anchored. 

Morgenroth  and  I  have  made  these  relations  more  easily  com- 
prehensible by  means  of  the  following  assumptions  concerning  the 
constitution  of  the  immune  body  and  complement. 

We  believe  it  necessary  to  assume  that  the  immune  body  possesses 
two  kinds  of  haptophore  groups,  one  of  high  affinity  which  combines 
with  a  corresponding  receptor  group  of  the  red  blood-cell  or  bacterium; 
the  other  a  group  of  less  affinity  which  combines  with  the  complement 
exerting  the  deleterious  action  on  the  cell.  Hence  the  immune  body 
is  a  kind  of  intermediate  element  which  links  complement  and  red 
blood -cells.  In  order  to  denote  this  function  I  have  proposed  the 
name  "  amboceptor, "  which  is  to  express  this  two-sided  grasping 
power. 

According  to  our  conception  the  complement  possesses  a  con- 
stitution analogous  to  that  of  the  toxins.  Thus  it  possesses  a  hapto- 
phore group  which  effects  the  specific  combination  with  the  ambo- 
ceptor.  The  presence  of  this  is  confirmed  by  the  existence  of  analogues 
of  antitoxins,  namely,  corresponding  anticomplements.  Besides  this 
the  complement  possesses  a  second  group,  the  cause  of  the  injurious 
action,  which  is  analogous  to  the  toxophore  group  of  the  toxins. 
In  view  of  the  properties  of  this  group,  partly  toxic,  partly  ferment- 
like,  I  have  decided  to  name  it  the  "zymotoxic"  group.  If  one  cares 


THE   PROTECTIVE  SUBSTANCES  OF  THE   BLOOD.  381 

to  illustrate  the  action  of  the  two  components  by  means  of  a  crude 
comparison,  the  action  of  gun  and  cartridge  may  be  taken.  The 
complement  in  itself  is  harmless,  like  a  cartridge,  whicn  only  acquires 
destructive  power  by  being  introduced  into  the  gun.  In  like  manner 
only  by  the  exclusive  mediation  of  the  amboceptor  is  the  injurious 
action  of  the  complement  called  forth  and  transmitted  to  certain 
particular  elements. 

In  opposition  to  this  conception  Bordet  maintains  the  view  that 
complement  and  immune  body  do  not  combine  as  we  believe,  but 
that  the *en trance  of  the  immune  body  into  the  cell  substance  exerts 
a  specific  injury  to  the  latter,  an  injury  which  manifests  itself  by 
the  fact  that  now  the  cells  succumb  to  the  action  of  the  simple  pro- 
tective substance  present  in  blood  serum,  namely,  Buchner's  "alexin." 

In  other  words,  by  means  of  the  immune  substances  the  blood- 
cells  are  made  susceptible,  "sensitized,"  to  the  action  of  the  alexin. 
In  conformity  with  this  Bordet  terms  our  immune  body  or  amboceptor 
the  "  substance  sensibilatrice"  and  our  complement  the  alexin. 

Although  this  view  is  also  shared  by  Buchner,  there  are  many 
reasons  why  I  cannot  accept  it,  especially  in  view  of  the  observation 
made  by  M.  Neisser  and  F.  Wechsberg  concerning  the  peculiar  phe- 
nomenon of  deflection  of  complement  through  an  excess  of  immune 
body.  To  begin  it  is  absolutely  impossible  to  picture  to  one's  sell  the 
nature  of  this  sensitization.  If  Bordet  believes  that  the  sensitizer  acts 
after  the  manner  of  a  safety-key  which,  when  introduced  into  a  par- 
ticular lock,  makes  the  introduction  of  a  second  key  possible,  I  must 
say  that  I  cannot  understand  this  comparison.  It  can  positively  be 
proven  that  the  red  blood-cell  possesses  no  complementophile  groups, 
since  neither  in  the  normal  state  nor  after  death  does  it  lay  hold 
of  complement.  The  living  blood-cell,  as  well  as  that  killed  by 
heating,  however,  through  the  occupation  with  the  immune  body, 
acquires  the  property  to  anchor  complement.  It  surely  is  much  more 
natural  to  believe  that  the  immune  body  itself,  the  amboceptor,  is 
the  carrier  of  the  group  which  binds  the  complement,  than  to  assume 
that  new  complementophile  groups  arise  owing  to  the  action  of  the 
sensitizer.  Finally,  one  can  conceive  of  such  a  process  in  a  living 
cell,  one  therefore  capable  of  alteration,  but  in  the  case  of  dead  cells 
which  have  been  treated  by  heat  or  all  sorts  of  chemicals,  in  the  case 
of  stabilized  albumin  as  one  might  say,  this  assumption  cannot  be 
allowed. 

Bordet 's  assumption  furthermore  does  not  explain  the  fact  that 


382  COLLECTED  STUDIES  IN  IMMUNITY. 

an  immune  body  derived  from  a  particular  species  is  most  surely 
activated  by  the  serum  derived  from  the  same  species.  From  the 
standpoint  of  Bordet's  theory  it  would  be  most  puzzling  to  under- 
stand why  an  anthrax  immune  body  derived  from  a  sheep  should 
sensitize  the  bacilli  against  just  the  sheep  alexin,  one  derived  from  a 
rabbit  against  just  the  rabbit  alexin.  From  the  standpoint  of  the 
amboceptor  theory,  however,  such  a  phenomenon  does  not  offer  the 
least  difficulty,  since  it  is  natural  that  the  amboceptors  circulating 
in  every  animal  species  are  fitted  to  their  own  complements. 

I  wish  to  mention  still  one  more  point  which  plays  a  great  role 
in'  Bordet's  views.  Bordet  assumes  that  the  alexin  is  a  simple  [ein- 
heitlich]  substance,  whereas  I  maintain  that  there  is  a  plurality  of 
complements.  Some  very  interesting  experiments  have  recently 
been  published  by  Bordet  which  appeared  to  support  the  Unitarian 
view. 

He  first  determined  that  a  certain  serum,  e.g.  guinea-pig  serum, 
was  able  to  activate  two  different  immune  bodies,  e.g.,  a  cholera- 
immune  body  and  a  hsemolytic  immune  body.  To  this  guinea-pig 
serum  Bordet  added  sensitized  blood-cells,  i.e.,  blood-cells  eager 
to  take  up,  and  susceptible  to  complement.  If  now  he  waited  until 
haemolysis  had  begun,  he  found  that  the  guinea-pig  serum  had  lost 
its  property  to  dissolve  sensitized  cholera  vibrios.  The  same  thing 
occurred  if  he  reversed  the  experiment. 

Although  it  was  easy  to  confirm  the  experiment  of  this  distin- 
guished investigator,  I  found  it  impossible  to  accept  Bordet's  con- 
clusions. This  experiment  is  only  then  positive  proof  for  a  simple 
alexin  (in  this  case  for  the  identity  of  bacteriolytic  and  haBmolytic 
alexin)  if  it  can  be  shown  that  the  two  immune  bodies  in  question 
are  acted  on  by  only  a  single  complementophile  group  and  not  by  a 
plurality  of  such  groups.  Previous  investigations,  however,  have 
shown  that  the  immune  sera  artificially  produced  are  not  simple 
in  character  but  are  made  up  of  a  number  of  different  amboceptors 
possessing  different  complementophile  groups. 

Nevertheless  I  consider  Bordet's  experiments  so  important  that  I 
have  once  more  had  this  question  thoroughly  studied  by  Dr.  Sachs 
and  Dr.  Morgenroth.  These  gentlemen  were  able  to  establish 
positive  proof  for  the  existence  of  different  complements.  Dr. 
Sachs,  for  instance,  studied  these  conditions  in  goat  serum,  employ- 
ing for  the  purpose  five  different  combinations  of  immune  body, 
each  of  which  could  be  complemented  by  goat  serum.  If  goat  serum 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  383 

contained  only  a  single  complement,  the  course  of  the  five  series  of 
tests  should  have  been  identical  when  the  complement  was  affected. 
It  was  found  on  the  contrary  that  under  the  influence  of  digestion, 
for  example,  one  completion  remained  intact,  while  four  others  dis- 
appeared. By  means  of  absorption  further  analogous  differences 
were  manifested  which  made  the  assumption  certain  that  in  this  case 
four  different  complements  come  into  action.  Since  these  results 
positively  prove  the  existence  of  a  plurality  of  complements  I  think 
it  will  be  unnecessary  here  to  bring  forward  additional  evidence  in 
support  of  this. 

A  resume  of  these  observations  confirms  my  view  that  the  mech- 
anism of  haemolysis  and  bacteriolysis  is  most  easily  explained  by 
the  amboceptor  theory. 

So  far  as  the  orgin  of  the  two  components  which  take  part  in 
this  reaction  are  concerned  there  is  not  the  least  doubt  that  they 
are  of  cellular  origin. 

I  assume  that,  in  addition  to  the  ordinary  receptors  which  serve 
to  take  up  relatively  simple  substances,  the  cells  contain  higher 
kinds  of  receptors  designed  to  take  up  large-moleculed  albuminous 
substances,  as,  for  example,  the  contents  of  living  cells.  In  this 
case,  however,  the  fixation  or  anchoring  of  the  molecule  constitutes 
only  a  prerequisite  for  the  cell's  nutrition.  Such  a  giant  molecule 
in  its  natural  state  is  useless  for  the  nutrition  of  the  cell  and  can 
be  utilized  only  after  it  has  been  broken  down  into  smaller  constit- 
uents by  fermentative  processes.  This  will  be  accomplished  most 
readily  if  the  grasping  group  of  the  protoplasm  is  also  the  carrier 
of  one  or  several  fermentative  groups  which  will  immediately  come 
into  close  relation  with  the  molecule  to  be  assimilated.  It  seems 
as  though  the  economy  of  cell  life  finds  it  advantageous  for  the  re- 
quired fermentative  groups  to  come  into  action  only  temporarily, 
perhaps  only  in  case  of  need.  This  purpose  is  effected  most  simply 
if  the  grasping  group  possesses  another  haptophore  group  which  can 
anchor  the  ferment-like  substances  present  in  the  serum,  the  comple- 
ments. Hence  such  a  receptor  of  the  higher  order  possesses  two  hapto- 
phore groups  of  which  one  anchors  the  foodstuff,  while  the  other  is 
complementophile.  It  is  obvious  that  when,  as  a  result  of  immuniza- 
tion, such  receptors  reach  the  blood,  they  will  exhibit  the  properties 
which  we  have  found  to  belong  to  the  receptor  type. 

In  regard  to  the  second  constituent,  the  complements,  we  shall 
not  err  if  we  regard  these  as  simple  cell  secretions,  designed  to  serve 


384  COLLECTED  STUDIES  IN   IMMUNITY. 

internal  metabolism.  In  accordance  with  the  conception  of  Metch- 
nikoff  we  must  for  the  present  believe  that  the  leucocytes  are  pri- 
marily concerned  in  their  production. 

From  these  points  of  view  the  organism's  immunity  reaction  loses 
the  mysterious  character  which  it  would  have  if  the  protective  sub- 
stances artificially  produced  represented  a  constituent  originally  for- 
eign to  the  organism  and  to  its  physiological  economy. 

But  we  have  seen  that  immunity  represents  nothing  more  than 
a  phase  of  the  general  physiology  of  nutrition,  a  view  in  which  I 
agree  entirely  with  that  distinguished  investigator  Metchnikoff. 
Phenomena  entirely  analogous  to  those  of  the  formation  of  anti- 
bodies are  constantly  occurring  in  the  economy  of  normal  metabolism, 
in  all  kinds  of  cells  in  the  organism  the  absorption  of  foodstuffs,  or 
of  products  of  intermediate  metabolism,  can  lead  to  the  formation  or 
the  thrus ting-off  of  receptors.  Considering  the  large  number  of  organs 
and  the  manifold  chemistry  of  their  cells  it  need  not  be  surprising 
that  the  blood,  which  is  representative  of  all  the  tissues,  contains 
an  innumerable  number  of  such  thrust-off  receptors.  To  these  I 
have  given  the  collective  name  of  "  hap  tins."  Only  in  recent  years, 
thanks  to  these  very  theoretical  considerations,  have  we  reached  a 
point  where  we  can  get  some  idea  of  this  enormous  multiplicity. 

In  addition  to  the  true  ferments  and  those  ferment-like  sub- 
stances, the  complements,  already  mentioned,  the  blood  normally 
contains  a  number  of  substances  which  act  specifically  against  cer- 
tain substances  present  in  solution. 

Chief  among  these  I  may  mention  the  normal  antitoxins,  and  as 
examples  of  these  the  diphtheria  antitoxin  and  antitetanolysin  of 
normal  horse  serum,  the  antistaphylotoxin  of  normal  human  serum, 
and  the  anticrotin  of  pig  serum.  Next  come  the  antiferments, 
such  as  antirennin,  antithrombase,  anticynarase,  and  others.  We 
also  normally  find  substances  which  prevent  the  action  of  specific 
haBmolysins  and  bacteriolysins,  being  directed  in  one  case  against 
the  amboceptor,  in  another  against  the  complement.  For  example^ 
in  goat  blood  I  discovered  an  an ti amboceptor  which  was  directed 
against  a  goat-blood  hsemolysin  obtained  in  accordance  with  Bordet's 
procedure.  In  the  blood  of  one  animal  species  P.  Muller  of  Graz 
found  antibodies  directed  against  certain  complements  of  other 
species  of  animals,  and  which  may,  therefore,  be  termed  normal 
anticomplements. 

Of  still  greater  interest,  however,  are  those  haptins  which  are 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  385 

directed  against  living  cells  of  all  kinds,  thus,  against  vegetable 
cells,  such  as  bacteria,  and  against  animal  cells,  such  as  red  blood- 
ceJls,  leucocytes,  spermatozoa,  epithelia,  and  others.  The  haptins 
which  are  so  antagonistic  to  cells  are  divisible  into  two  large  groups: 
(1)  the  agglutinins,  which  cause  the  bacteria  or  other  cells  to  stick 
together,  and  which  through  the  researches  of  Gruber,  Durham, 
and  Widal  have  attained  such  great  diagnostic  significance;  (2) 
the  bactericidal  or  cytotoxic  substances,  and  these  are  intimately 
related  to  natural  immunity.  In  case  the  substances  not  only  kill 
but  also  exert  a  solvent  action  we  call  them  lysins,  and  speak  of 
hsemolysins,  bacteriolysins,  etc.  Thus  a  certain  blood  serum,  e.g. 
dog  serum,  will  simultaneously  exert  antitoxic,  antifermentative, 
agglutinating,  bacteriolytic,  and  cytotoxic  effects  against  the  appro- 
priate substances.  If  we  consider  one  of  these  functions  by  itself, 
e.g.,  the  agglutinating  function  of  a  certain  serum,  we  shall  be  met 
with  the  question  whether  or  not  this  property  is  due  to  one  simple 
substance,  the  agglutinin.  Numerous  experiments  have  shown  that 
this  is  not  so,  but  that  in  this  precipitating  process  just  exactly  as 
many  different  agglutinins  take  part  as  there  are  present  different 
agglutinable  substances.  It  is  easy  to  demonstrate  this  plurality 
by  means  of  the  principle  of  specific  union  introduced  by  me. 

If,  for  example,  a  certain  serum  is  able  to  agglutinate  two  varieties  of  blood- 
cells,  say  rabbit  and  pigeon  blood-cells,  and  two  kinds  of  bacteria,  as  cholera 
and  typhoid,  it  should  be  tound,  in  case  this  plural  effect  were  produced  by 
a  single  simple  agglutinin,  that  absorption  by  one  of  these  elements,  e.g.  the 
cholera  vibrios,  would  remove  the  other  three  actions  also.  As  a  matter  of 
fact,  however,  the  serum  which  has  been  shaken  with  cholera  vibrios,  while 
it  will  no  longer  agglutinate  cholera  vibrios,  is  still  able  to  produce  agglutina- 
tion in  the  other  three  elements,  and  vice  versa.  In  this  case,  therefore,  tour 
different  agglutinations  take  part. 

Results  entirely  analogous  to  these  are  obtained  if  the  other 
functionating  groups  contained  in  blood,  e.g.  the  antitoxic,  bacterio- 
lytic, etc.,  are  examined  in  a  corresponding  manner.  These  facts 
confirm  the  pluralistic  view  first  maintained  by  me,  according  to 
which  every  blood  serum  contains  many  hundreds,  or  even  thou- 
sands, of  effective  haptins.  All  of  these,  with  the  exception,  per- 
haps, of  ferments  and  complements,  owe  their  origin  to  an  excessive 
assimilative  metabolism.  Their  peculiar  action  on  certain  substances 
foreign  to  the  body  may  be  regarded  as  due  to  an  incidental 
meeting.  To  a  large  extent,  therefore;  they  are  to  be  looked  upon 


386  COLLECTED  STUDIES  IN  IMMUNITY. 

as  luxuries  which  are  not  in  themselves  of  any  significance  for  the 
life  of  the  organism.  Of  what  use  is  it  to  a  person  or  to  an  animal 
to  have  circulating  in  his  blood  a  great  variety  of  substances 
directed  against  heterogeneous  materials  which  under  normal  cir- 
cumstances never  come  into  account,  and  which  at  the  most  are 
brought  into  relation  with  these  substances  only  by  the  experi- 
menter? Of  what  use  is  it  to  a  goat  to  have  in  its  blood  certain 
substances  which  are  directed  against  the  red  blood-cells  or  the 
spermatozoa  of  other  animals,  since  these  do  not  normally  get  into 
the  circulation?  Furthermore  every  experimenter  finds  that  the 
blood  serum  is  subject  to  constant  change  in  most  of  its  haptins,  a 
fact  which  argues  strongly  against  the  assumption  that  all  of  these 
substances  in  a  free  state  play  an  important  or  even  necessary  role 
in  the  organism. 

I  cannot  and  do  not  deny  that  with  such  a  superabundance  of 
combinations  in  every  serum  substances  will  also  be  present  which 
either  by  themselves  or  in  conjunction  with  complements  are  able 
to  destroy  invading  injurious  bodies,  especially  bacteria.  These 
substances  then  may  be  regarded  as  acting  as  defensive  agents.  In 
spite  of  this,  however,  I  believe  it  is  wrong  to  group  this  most  com- 
plex system  of  haptins  under  the  collective  name  alexin,  because 
this  leads  to  an  incorrect  Unitarian  view  which  cannot  help  scientific 
progress.  These  remarks  are  in  no  way  intended  to  detract  from 
the  very  valuable  work  of  Buchner;  his  study  on  alexins,  viewed 
in  the  light  of  that  time  and  according  to  the  then  state  of  science, 
must  be  regarded  as  a  masterpiece  which  has  been  of  enormous  value 
in  the  development  of  this  subject. 

Still  another  difference  of  opinion  existing  between  Buchner 
and  myself  concerns  the  bactericidal  and  hsemolytic  power  of  nor- 
mal blood  serum,  and  these  properties  Buchner  again  ascribes  to 
the  action  of  his  alexin  conceived  as  a  simple  substance.  In  oppo- 
sition to  this  I  have  demonstrated  that  the  conditions  in  normal 
haemolysins  are  exactly  the  same  as  in  the  artificial  hsemolysins, 
for  here  again  two  different  components  act  together:  one  of  them 
is  thermostable  while  the  other  corresponds  to  the  complements. 
This  fact  has  been  confirmed  by  a  large  number  of  observers,  among 
whom  I  may  mention  v.  Dungern,  Moxter,  London,  P.  Muller,  Meltzer. 
All  these  authors,  like  myself,  have  come  to  the  conclusion  that  the 
thermostable  substance  necessary  for  the  lytic  process  corresponds 
in  every  way  to  the  artificially  produced  immune  bodies  or  ambo- 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  387 

ceptors.  The  haemolysins  occurring  naturally  and  those  artificially 
produced  manifest  their  action  according  to  exactly  the  same  mechan- 
ism. According  to  the  observations  of  Pfeiffer  and  of  Moxter,  as 
well  as  to  certain  experiments  of  Wechsberg  and  M.  Neisser,  still 
to  be  published,  the  same  holds  true  for  the  bactericidal  substances. 

Against  this  view  Buchner,  while  in  general  he  confirms  our  find- 
ings of  fact,  maintains  that  the  thermostable  substances  of  normal 
sera  are  not  analogous  to  the  immune  bodies,  but  are  something 
apart  by  themselves.  He  therefore  gives  them  a  distinct  name, 
"  Hilfskorper "  [= aiding  body].  Such  a  separation  of  the  con- 
nection between  the  physiological  and  the  pathological  is  opposed 
to  the  teachings  of  Virchow.  Aside  from  this,  however,  I  regard 
the  proof  which  Buchner  advances  for  placing  these  "  Hilfskorper  " 
by  themselves  as  insufficient.  It  is  entirely  negative  and  consists 
in  this,  that,  according  to  Buchner,  proof  has  not  been  offered  that 
in  normal  haemolysis  "a  "  Hilfskorper  "  does  not  always  come  into 
action.  Against  this  I  should  like  to  point  out  that,  in  the  very 
large  number  of  cases  of  normal  haemolysis  studied  during  the  past 
years  by  myself  and  fellow  workers,  we  have  always  succeeded  in 
discovering  the  amboceptor  effecting  the  action.  At  times,  of  course, 
this  required  a  great  deal  of  labor  and  trying  all  sorts  of  sources 
for  complement.  Experiments  like  those  recently  published  by 
Buchner,  in  which  only  one  combination  chosen  at  random  from 
the  many  possible  ones  is  employed,  do  not  argue  against  the  pres- 
ence of  amboceptors  in  case  the  experiment  results  negatively,  for 
no  one  versed  in  this  subject  would  assume  that  every  amboceptor 
must  find  a  fitting  complement  in  every  serum  used.  Hence  Buchner 
does  not  furnish  any  proof  that  hamolysis  can  be  produced  by  the 
alexin  alone. 

In  connection  with  this  I  should  like  to  call  attention  to  the  fact 
that  the  alexin  or  complement  action  possessed  by  normal  serum  is 
due  to  a  plurality  of  substances,  not  to  a  single  one.  Each  comple- 
ment by  itself  is  harmless,  for  only  through  the  intervention  of  the 
amboceptor  is  its  injurious  action  carried  over  to  certain  tissues. 
When  this  occurs,  however,  the  action  is  the  same  on  its  own  as  on 
foreign  tissues.  It  is  surprising  to  watch  how  guinea-pig  blood-cells 
which  have  been  loaded  or  sensitized  with  certain  amboceptors  at 
once  dissolve  if  their  own  serum  is  added,  this  serum  now  acting  as 
a  deadly  poison.  There  is  very  little  ground,  therefore,  to  regard  the 
complements  as  playing  the  role  of  defenders  against  foreign  invaders. 


388  COLLECTED  STUDIES  IN   IMMUNITY. 

That  they  appear  to  play  this  role  is  due  to  the  action  of  what  I  have 
termed  the  "horror  autotoxicus,"  which  prevents  the  production 
within  the  organism  of  amboceptors  directed  against  its  own  tissues. 

In  this  "horror  autotoxicus  "  we  are  dealing  with  a  well-adapted 
regulatory  contrivance  which  it  may  be  well  to  discuss  briefly.  The 
investigations  of  numerous  authors  have  shown  that  by  injecting 
animals  with  any  kind  of  foreign  cell  material  cytotoxic  substances 
can  be  produced  directed  exactly  against  the  material  used  for  im- 
munization. Thus  if  a  dog  is  immunized  with  an  emulsion  of  goose 
brain,  it  will  be  found  that  the  dog's  serum  will  be  highly  toxic  only 
for  geese,  killing  these  animals  with  cerebral  symptoms.  In  the  same 
way  we  can  produce  other  poisons,  hepatotoxins,  nephrotoxins,  etc., 
each  of  which  acts  only  on  a  certain  organ  of  a  particular  species. 
In  human  pathology,  however,  we  must  consider  the  absorption  of 
the  body's  own  constituents  and  not  of  those  of  other  bodies.  The 
former  may  occur  under  many  conditions;  for  example,  in  hemorrhages 
into  the  body  cavities,  in  the  absorption  of  lymph-gland  tumors,  in 
the  febrile  waste  of  body  parenchyma.  It  would  be  dysteleological 
to  the  highest  degree  if  under  these  circumstances  poisons  against 
the  body's  own  parenchyma,  auto  toxins,  were  to  arise.  I  have 
attempted  to  solve  this  question  by  injecting  goats  with  the  blood  of 
other  goats.  The  sera  of  animals  so  treated  did  not  dissolve  their 
own  blood-cells,  but  dissolved  those  of  other  goats.  Hence  it  did 
not  contain  an  autotoxin,  but  an  "isotoxin,"  in  conformity  with  the 
law  to  which  I  give  the  name  "horror  autotoxicus." 

I  believe  that  the  isotoxins  may  perhaps  come  to  play  an  im- 
portant role  in  diagnosis  and  pathology.  In  the  serum  of  dogs  in 
which  he  had  produced  a  chromium  nephritis,  Metchnikoff  found 
that  an  isonephro toxin  had  developed,  for  when  this  serum  was 
injected  into  normal  dogs  it  produced  a  nephritis.  It  is  more  than 
probable  that  in  man  also  the  greatest  variety  of  isotoxins  is  formed. 
In  the  case  of  the  blood  this  has  already  been  positively  demonstrated 
by  a  number  of  authors,  such  as  Landsteiner,  Ascoli,  etc. 

With  the  exception  of  the  red  blood  corpuscles  we  cannot,  of 
course,  undertake  any  studies  in  man  concerning  the  isotoxins  of 
the  parenchyma.  Many  considerations,  however,  indicate  that  it 
will  be  possible  to  carry  out  these  experiments  on  monkeys  and  so 
gain  a  new  foundation  for  pathology  and  therapy  in  man. 

The  number  of  combinations  present  in  the  blood  serum  and 
making  up  the  ever-changing  haptin  apparatus  is  infinitely  great. 


THE  PROTECTIVE  SUBSTANCES  OF  THE  BLOOD.  389 

Of  these  especially  the  substances  of  the  amboceptor  type  are  in 
most  intimate  relationship  to  the  processes  of  natural  immunity,  for 
it  is  they  which,  in  conjunction  with  the  complement,  effect  the  de- 
struction of  the  injurious  bacteria.  Hence  if  there  is  a  loss  of  natural 
immunity,  it  will  next  be  necessary  to  inquire  whether  there  is  a  lack 
of  complement  or  of  amboceptor. 

I  am  convinced  that  these  haptin  studies  open  up  a  new  and 
important  field  of  biological  investigation  and  will  add  to  our  knowl- 
edge concerning  the  process  of  assimilation.  Clinically  they  should 
be  of  even  greater  importance.  Since  I  am  not  in  the  position  to 
make  such  chemical  investigations  on  an  abundance  of  material,  I 
have  thought  it  my  duty  to  clearly  define  my  point  of  view,  thus 
furnishing  to  others  the  basis  for  a  proper  study  of  this  subject.  The 
significance  of  this  method  for  pathology  and  therapy  will  not  perhaps 
be  fully  realized  until  after  the  lapse  of  years. 


XXXIII.    THE    RECEPTOR  APPARATUS    OF  THE    RED 

BLOOD-CELLS.1 

By  Professor  Dr.  P.  EHRLICH. 

WE  know  of  a  large  number  of  agents  which  are  able  to  injure 
the  red  blood-cells  or  kill  them.  In  a  study  entitled  "  Zur  Physiologic 
und  Pathologic  der  rothen  Blutscheiben  "  (Charite  Annalen,  Vol.  10) 
I  have  shown  that  solution  of  red  blood-cells  is  brought  about  by 
all  agencies  (mechanical,  chemical,  or  thermic)  which  kill  proto- 
plasm. At  that  time  I  had  already  expressed  the  hypothesis  that 
the  erythrocytes  possessed  a  peculiar  protoplasm,  the  discoplasma, 
whose  chief  function  consists  in  preventing  the  escape  of  the  haemo- 
globin into  the  blood  plasma.  If  the  discoplasma  is  killed,  the  haemo- 
globin will  immediately  diffuse,  i.e.,  the  blood  becomes  laky.  This 
process  is  in  no  way  connected  with  conditions  of  osmotic  tension, 
for  in  many  blood  poisons,  such  as  digitoxin,  veratrin,  solan  in,  cor- 
rosive sublimate,  etc.,  this  destruction  takes  place  in  very  high  dilu- 
tions which  hardly  change  the  molecular  concentration  at  all. 

The  ordinary  blood  poisons,  and  they  are  very  numerous  (saponin 
bodies,  helvellic  acid,  aldehydes,  polyphenols,  etc.),  are  chemically 
clearly  defined  substances;  they  exert  their  deleterious  action  in 
exact  accordance  with  the  principles  which  we  have  already  studied 
in  connection  with  the  distribution  of  pharmacological  substances, 
such  as  alkaloids,  etc.  Recently,  however,  we  have  come  to  know 
another  group  of  blood  poisons  which  exert  their  injurious  action 
after  the  manner  of  toxins,  i.e.,  through  the  agency  of  special  hapto- 
phore  groups  which  fit  into  suitable  receptors.  All  of  these  sub- 
stances are  highly  complex  derivatives  of  living  animal  or  vegetable 

1  Reprint  from:  Schlussbetrachtungen ;  Erkrankungen  des  Blutes;  Noth- 
nagel's  Specielle  Pathologic  und  Therapie,  Vol.  VIII,  Vienna,  1901. 

390 


THE  RECEPTOR  APPARATUS  OF    THE  RED   BLOOD-CELLS.  391 

cells;  for  the  present  at  least  their  chemical  nature  is  unknown.     Into 
this  class,  to  mention  only  the  simplest  types,  belong  the  following: 

1.  Poisonous  phytalbumoses:  ricin,  abrin;  crotin,  phallin; 

2.  Bacterial  secretions:    tetanolysin   (Ehrlich,   Madsen),  staphylo- 
toxin  (van  de  Velde,  M.  Neisser,  and  F.  Wechsberg),  pyocyaneous 
poison    (Bulloch),    streptococcus    poison    (v.    Lingelsheim),    cholera 
poison,  and  probably  many  others. 

3.  Poisonous    animal    secretions,    especially    the    various    snake 
venoms. 

The  majority  of  these  substances,  especially  all  of  the  bacterial 
products,  produce  ordinary  ha3molysis.  In  contrast  to  this,  as 
Kobert  has  shown,  abrin  and  ricin  cause  a  rapid  clumping  of  the 
erythrocytes,  a  process  which  is  analogous  to  the  agglutinative  phe- 
nomena studied  by  Gruber,  Durham,  and  Widal.  However,  in  the 
case  of  the  poisonous  phytalbumoses  we  cannot  assume  that  there 
is  an  essential  difference  between  hamolysis  and  agglutinatin,  be- 
cause one  of  them,  crotin,  has  been  shown  by  Elfstrand  to  exert  a 
pure  agglutining  action  on  certain  species  of  blood  (sheep,  pig,  ox) 
and  a  pure  solvent  action  on  others  (rabbit).1 

Of  especial  importance,  however,  is  the  fact  that  all  these  poisons 
on  being  introduced  into  the  animal  body  produce  specific  antitox- 
ins (aritiricin,  antiabrin  (Ehrlich);  anticrotin  (Morgenroth) ;  anti- 
tetanolysin  (Madsen);  antileucocidin  (van  de  Velde).  In  view  of 
what  we  have  already  discussed  this  fact  alone  is  sufficient  to  ascribe 
to  these  substances  the  possession  of  a  haptophore  group  through 
which  they  exert  their  toxicity.  Furthermore,  just  like  the  true 
toxins,  they  possess  a  second  group  which  is  the  cause  of  the  toxic 
action.  As  Madsen  has  shown  in  the  case  of  tetanolysin,  and  M.  Neisser 
and  F.  Wechsberg  for  staphylolysin,  it  is  possible  to  change  these 
poisons  into  modifications  which  have  more  or  less  completely  lost 
their  toxicity  but  which  preserve  unchanged  the  properties  dependent 
on  the  possession  of  the  haptophore  group  (affinity  for  the  anti- 
body, production  of  immunity).  These  modifications,  first  recognized 


1  Even  ricin,  which  is  apparently  purely  agglutinating,  exerts  an  action  on 
the  discoplasma  which  causes  haemolysis.  In  the  ordinary  technique  of  the 
experiment  this  action  is  obscured  by  the  fact  that  in  the  agglutinated  masses 
the  conditions  are  very  unfavorable  for  diffusion.  If  these  conditions  are 
made  more  favorable  by  breaking  up  the  clumps  by  shaking,  one  can  easily 
observe  the  escape  of  the  haemoglobin. 


392  COLLECTED  STUDIES  IN    IMMUNITY. 

by  me  in  diphtheria  poisons,  depend  on  the  separate  destruction  of 
the  very  unstable  toxophore  group. 

In  passing  now  to  the  substances  contained  in  blood  plasma  I 
shall  discuss  first  the  agglutinins.  Even  normal  serum  frequently 
contains  substances  which  clump  certain  bacteria  and  erythrocytes. 
Although  at  first,  in  accordance  with  Buchner's  views,  one  single 
substance  was  made  responsible  for  the  different  actions,  I  believe 
that  at  present  the  pluralistic  standpoint  first  maintained  by  me  is 
generally  accepted.  The  plurality  of  normal  agglutinins  was  at  once 
proven  as  soon  as  my  principle  of  specific  combination  was  applied  to 
this  question,  as  was  done  by  Bordet  and  Malkow.  The  latter  showed 
that  if  goat  serum  which  agglutinates  the  erythrocytes  of  pigeon, 
man,  and  rabbit  is  shaken  with  the  red  cells  of  one  of  these  species, 
e.g.  pigeon,  it  will  be  found  that  the  centrifuged  fluid  still  contains 
the  two  other  agglutinins  unchanged,  whereas  the  agglutinin  for  pigeon 
blood  is  absent. 

These  substances  can  be  obtained  artificially  by  following  the 
procedure  of  Belfanti  and  Carbone,  who  injected  animals  with  con- 
siderable amounts  of  foreign  red  blood-cells  (blood-cell  immunization). 
They  are  readily  separated  from  the  hsemolysins  developing  simul- 
taneously by  heating  for  half  an  hour  to  56°  C.  As  a  result  of  this 
the  action  of  the  amboceptor  iysins  is  destroyed  while  the  agglutinins 
themselves  are  unaffected.  To  be  sure  if  the  temperature  is  increased 
to  70°  C.  it  is  possible  to  destroy  also  the  agglutinating  action.  In 
that  case,  however,  the  addition  of  normal  serum  no  longer  exerts  a 
reactivating  action.  From  this  it  follows  that  the  agglutinins  l  are 
not  of  such  complex  constitution  as  the  amboceptor  Iysins;  analogous 
to  the  toxins  they  contain  a  haptophore  group  and  a  zymophore 
which  causes  the  coagulation  process.  In  accordance  with  this  I 
believe  that  the  agglutinins  are  nothing  more  than  receptors  of  the 
second  order.2 

1  The  agglutinins  here  described,  in  contrast  to  ricin  and  abrin,  give  rise 
to  no  further  injurious  action  on  the  discoplasma. 

2  In  the  first  part  of  "Schlussbetrachtungen"  I  have  distinguished: 

1.  Receptors  of  the  first  order,  which  concern  themselves  with  the  assimilation 
of  simple  substances  (toxins,  ferments,  and  other  cell  secretions).     For  this 
purpose  a  single  haptophore  group  suffices.     When  thrust  off  into  the  blood 
in  consequence  of  the  introduction  of  toxins,  these  receptors  constitute  the 
antitoxins  (antiferments). 

2.  Receptors  of  the  second  order,  which  in  addition  to  the  haptophore  group 
possess  a  second  group  which  effects  the  coagulation.     After  they  have  been 


THE  RECEPTOR  APPARATUS  OF  THE  RED  BLOOD-CELLS.  393 


m 


FIG.  1. — THE  VARIOUS  TYPES  OF  RECEPTORS  ACCORDING  TO  EHRLICH. 

I.  Receptors  of  the  First  Order. — This  type  is  pictured  in  a.  The  portion  e 
represents  the  haptophore  group,  whilst  6  represents  a  toxin  molecule, 
which  possesses  a  haptophore  group  c  and  a  toxophore  group  d.  This 
represents  the  union  of  toxin  and  antitoxin,  or  ferment  and  antifer- 
ment,  the  union  between  antibody  and  the  toxin  or  ferment  being  direct. 
II.  Receptors  of  the  Second  Order  are  pictured  in  c.  Here  e  represents  the 
haptophore  group,  and  d  the  zymophore  group  of  the  receptor,  /  being 
the  food  molecule  with  which  this  receptor  combines.  Such  receptors 
are  possessed  by  agglutinins  and  precipitins.  It  is  to  be  noted  that 
the  zymophore  group  is  an  integral  part  of  the  receptor. 

III.  Receptors  of  the  Third  Order  are  pictured  in  III,  e  being  the  haptophore 
group  and  g  the  complementophile  group  of  the  receptor.  The  com- 
plement k  possesses  a  haptophore  group  h  and  zymotoxic  group  2; 
whilst  /  represents  the  food  molecule  which  has  become  linked  to  the 
receptor.  Such  receptors  are  found  in  hapmolysins,  bacteriolysins,  and 
other  cytolysins,  the  union  with  these  cellular  elements  being  effected 
by  the  amboceptor  (a  thrust-off  receptor  of  this  order).  It  is  to  be 
noted  that  the  digesting  body,  the  complement,  is  distinct  from  the 
receptor,  a  point  in  which  these  receptors  therefore  differ  from  those 
of  the  preceding  order. 


394  COLLECTED  STUDIES  IN   IMMUNITY. 

Next  we  come  to  the  very  important  substances  in  serum  which 
-cause  haemolysis.  I  have  previously  dwelt  in  detail  on  the  fact  that 
in  this  the  action  is  always  due  to  amboceptors  which  attract  both 
blood-cells  and  complement.  Hence  I  may  limit  myself  at  this  time 
to  some  supplementary  remarks.  It  has  long  been  known  that  the 
blood  serum  of  one  species  injures  and  dissolves  the  erythrocytes  of 
other  animal  species.  This  is  the  case  not  only  in  distantly  related 
types,  such  as  fish  and  mammals,  but,  as  was  shown  by  therapeutic 
blood  transfusions,  occurs  also  in  comparatively  near  relatives. 
Buchner  was  the  first  to  appreciate  the  significance  of  this  phenomenon, 
and  assumed  that  the  serum  contained  a  substance  innocuous  for  its 
own  body  but  acting  destructively  on  foreign  elements  (bacteria  and 
Mood-cells) .  This  substance  he  therefore  terms  alexin.  Not  until, 
in  later  years,  the  mechanism  of  artificially  produced  lysins  became 
<;lear  was  this  Unitarian  view  shown  to  be  untenable.  First  it  was 
found  that  the  lysins  contained  in  normal  blood  are  not  simple  in 
nature,  but  are  composed  just  like  those  artificially  produced,  of 
two  components,  the  amboceptor  and  the  fitting  complement.  Further- 
more, corresponding  to  the  results  in  the  case  of  agglutinins,  and  by 
means  of  the  same  methods,  it  was  found  that  a  given  serum  can  con- 
tain a  large  number  of  different  amboceptor  lysins.  If  a  certain 
,serum  (e.g.  dog  serum)  dissolves  the  erythrocytes  of  different  species, 
the  specific  combining  method  has  shown  that  this  property  is  due 
to  the  presence  of  different  amboceptors,  each  of  which  is  related 
to  only  one  of  these  species  of  blood-cells.  In  fact  it  even  seems  as 
if  different  complements  may  correspond  to  these  amboceptors. 

In  view  of  what  has  been  said  we  are  fortunately  able  to  regard 
these  different  agents  which  injure  the  blood  from  a  common  point 
of  view.  Whether  we  are  dealing  with  vegetable  or  animal  prod- 
ucts, whether  with  lysins  or  agglutinins,  whether  with  substances 
of  toxin-like  nature  or  of  the  complex  amboceptor  type, — in  all  of 
these  cases  the  prerequisite  and  cause  of  this  poisonous  action  is  the 

thrust  off  into  the  blood  they  constitute  agglutinins  and  precipitins.  The 
toxins  also  are  to  be  regarded  as  receptors  of  the  second  order  thrust  off  by 
bacteria. 

3.  Receptors  of  the  third  order,  which  possess  two  haptophore  groups,  one 
of  which  effects  the  union  with  the  foodstuff,  whereas  the  other  lays  hold  on 
certain  substances  circulating  in  the  blood  plasma,  the  complements,  which 
cause  ferment-like  actions.  After  they  are  thrust  off  these  receptors  con- 
.situte  the  "amboceptors." 


THE   RECEPTOR   APPARATUS  OF    THE   RED   BLOOD-CELLS    395 

same,  namely,  the  presence  of  suitable  receptors  on  the  blood-discs,  i.e., 
receptors  which  fit  the  haptophore  groups  of  the  toxin  or  the  corre- 
sponding groups  of  the  amboceptor.  This  view,  already  generally 
accepted  for  the  toxin  poisonings,  is  supported  by  considerations  of 
two  kinds.  First  is  the  positive  proof  in  the  case  of  the  manifold 
blood  poisons,  that  their  injurious  action  is  always  preceded  by  the 
anchoring  of  the  poison  to  the  blood-cell.  Only  such  species  of 
blood-cells  are  susceptible  to  a  certain  haemolysin  which  are  able 
to  anchor  the  same.  This  has  been  confirmed  again  and  again  in 
the  case  of  amboceptor  lysins.  Conversely,  therefore,  there  is  the 
closest  connection  between  natural  immunity  and  absence  of  receptors. 
That  the  fixation  of  the  poisons  is  not  due  to  mechanical  effects, 
such  as  surface  attraction,  but  to  a  true  chemical  process,  is  at  once 
shown  by  the  strict  specificity  which  obtains.  This  is  observed 
especially  in  the  amboceptor  lysins  produced  artificially.  This 
specificity  is  in  marked  contrast  to  the  many-sided  and  non-selective 
action  of  surface  attraction  (charcoal,  etc.).  The  second  point 
which  supports  the  above  view  is  the  fact  that  the  action  of  a 
certain  poison,  and  only  of  this  one,  is  inhibited  by  the  correspond- 
ing antitoxin.  According  to  my  views,  the  action  of  antitoxins  is 
explained  by  assuming  that  they  occupy  the  haptophore  groups  of 
the  toxin  molecule  and  so  prevent  these  from  combining  with  the 
receptors  of  the  tissues.  It  is  quite  incomprehensible  to  me  how 
the  specificity  of  the  antitoxins  can  more  easily  be  explained  on 
the  basis  of  the  mechanical  conception. 

This  brings  us  to  a  very  important  point,  namely,  the  surprising 
plurality  of  receptors.  Even  in  the  blood  poisons  each  antiserum 
protects  only  against  the  substance  through  which  it  was  produced 
by  immunization.  This  law  of  specificity,  which  has  so  repeatedly 
been  confirmed  in  the  infectious  diseases,  is  thus  seen  to  apply  here 
without  any  change.  Antiricin  serum  protects  the  blood-cells  only 
against  ricin,  antitetanolysin  only  against  tetanolysin,  every  anti- 
amboceptor  only  against  a  corresponding  amboceptor. 

Hence  in  every  species  of  blood-cell  we  shall  have  to  assume 
the  existence  of  as  many  different  kinds  of  receptors  as  there  are 
poisons.  This  is  obviously  a  very  large  number.  Thus  if  the  blood- 
cells  of  rabbits  are  injured  by  ricin,  crotin,  abrin,  phallin,  by  the 
most  diverse  products  of  bacterial  metabolism,  and  by  a  large  num- 
ber of  sera  of  other  species,  we  shall  have  to  assume  a  certain  recep- 
tor (ricin  receptor,  etc.)  for  each  case.  Almost  every  day,  however, 


396  COLLECTED  STUDIES  IN   IMMUNITY 

we  are  coming  to  know  more  such  blood  poisons;  the  number  of 
different  receptors  which  we  can  determine,  therefore,  continues 
to  increase. 

In  this  connection  I  should  like  to  present  the  results  which 
Dr.  Morgenroth  and  I  have  obtained  in  attempting  to  produce  auto- 
lysins  by  immunizing  goats  with  blood  from  the  same  species  instead 
of  blood  from  foreign  species.  In  only  one  single  instance  were  we 
successful,  i.e.,  in  obtaining  a  solution  of  the  animal's  own  blood- 
cells.  In  all  other  cases  we  obtained  merely  an  isolysin,  which  dis- 
solved the  blood-cells  of  other  goats  but  not  those  of  the  goat  immu- 
nized. If  the  blood  of  a  large  number  of  goats  is  tested  with  a  par- 
ticular isolysin,  it  would  be  found  that  of  some  goats  the  blood  is 
highly  susceptible,  of  others  it  is  feebly  susceptible,  and  of  still  others 
the  blood  is  not  at  all  susceptible.  In  the  case  of  the  susceptible 
bloods  it  can  be  shown  that  the  isolysin  consists  of  the  arnboceptor 
which  is  anchored,  plus  a  complement  of  normal  goat  serum.  In 
course  of  time  we  have  produced  thirteen  such  isolytic  sera,  and  found 
to  our  surprise  that  they  all  differed  from  one  another,  i.e.,  that 
they  represented  different  isolysins.  Thus  the  first  serum  dissolved 
the  blood-cells  of  A  and  B;  a  second  serum  those  of  C  and  D;  a 
third  A  and  D,  etc.  By  means  of  this  one  experiment  we  have, 
therefore,  come  to  know  thirteen  different  lysins,  to  which,  of  course, 
a  similar  number  of  receptors  must  correspond.  It  was  fortunate 
for  us  that  in  the  blood-cells  of  an  animal  all  the  receptors  were  not 
present,  but  only  a  part  of  the  same,  for  it  was  only  owing  to  this 
fact  that  a  separation  of  the  different  kinds  was  possible. 

It  is  worthy  of  note  that  many  receptors  may  be  present  in  the 
blood-cells  in  relatively  large  amounts.  If  we  designate  as  the  single 
lethal  dose  (L.D.)  that  amount  of  a  certain  arnboceptor  which  when 
supplied  with  sufficient  complement  just  suffices  to  completely  dis- 
solve a  constant  amount  of  blood,  we  can,  by  employing  different 
amounts  of  amboceptor  solutions  inactivated  by  heat,  readily  deter- 
mine how  many  L.D.  can  be  anchored  by  the  amount  of  blood  in 
question.  As  a  result  of  this  it  has  been  found  that  in  some  cases 
only  just  the  single  L.D.  is  bound.  More  frequently  the  combining 
power  of  the  erythrocytes  is  much  higher,  so  that  two  to  ten  and 
even  fifty  times  the  L.D.  is  bound.  In  such  cases,  therefore,  we 
are  dealing  with  a  marked  excess  of  these  particular  receptors.  An 
analogous  case,  by  the  way,  has  long  been  known  as  a  result  of 
Wasseimann's  experiment  concerning  the  power  of  brain  substance 


THE  RECEPTOR  APPARATUS  OF    THE   RED   BLOOD-CELLS.  397 

to  bind  tetanus  poison.  In  virtue  of  such  an  excess  of  tetanus 
receptors,  the  brain  also  absorbs  a  considerable  multiple  of  the  L.D. 
Hence  in  test-tube  experiments  it  is  still  possible  to  neutralize  con- 
siderable quantities  of  poison  with  the  brain  of  a  guinea-pig  which 
has  died  of  tetanus. 

All  of  these  tacts  lead  to  the  conception  that  the  red  blood-cells 
possess  an  enormous  number  of  receptors  which  probably  belong 
to  hundreds  of  different  types.  Of  these,  again,  a  few  may  be  present 
in  relatively  large  quantities.  This  fact  is  surprising;  for  in  a  way 
it  is  opposed  to  the  view  held  until  now  concerning  the  function 
of  the  red  blood-cells.  It  is  inconceivable  that  the  simple  inter- 
change of  oxygen,  a  purely  chemical  function  of  the  haemoglobin, 
would  require  so  complex  an  arrangement  as  that  just  described. 
In  my  opinion,  therefore,  this  enormous  apparatus  indicates  that 
the  red  blood-cells  actually  exercise  properties  which  we  have  thus 
far  overlooked.  If  we  consider  that  the  receptors  in  general  serve 
to  take  up  foodstuffs,  or  in  some  cases  the  products  of  internal 
metabolism,  we  may  easily  assume  that  the  receptor  apparatus  of 
the  erythrocytes  fulfills  this  same  purpose.  Since,  however,  we 
know  that  the  vita  propria  of  the  blood-cells  is  very  limited,  we 
shall  have  to  assume  that  the  substances  taken  up  are  not  for  the 
blood-cells'  own  consumption,  but  are  designed  to  be  given  off  to 
other  organs.  The  red  blood-cells  may  therefore  be  regarded  as 
storage  reservoirs  in  the  sense  that  they  temporarily  take  up  the 
most  varied  substances  derived  from  the  food  or  from  the  internal 
metabolism,  provided  these  substances  are  supplied  with  haptophore 
groups.  I  may  be  permitted  to  call  attention  to  the  fact  that  the 
erythrocytes  contain  chiefly  receptors  ot  the  first  order,1  i.e.,  recep- 
tors which  take  up  substances  but  do  not  further  digest  them. 

After  these  explanations  I  feel  justified  in  believing  that  the 
study  of  receptors  has  opened  up  a  new  and  important  field  of  bio- 
logical investigation.  In  order  to  make  my  meaning  clearer  I  should 
like  to  quote  the  following  paragraph  from  Verworn  (Beitrage  zur 
Physiologic  des  central  N  erven-Systems,  I.  Thiel,  page  68)  in  which 
our  present  knowledge  is  reviewed:  "The  living  substance  of  every 
cell,  so  long  as  it  actually  is  living  and  manifests  vital  phenomena, 
is  constantly  decomposing  automatically  and  constantly  forming 
new  substances.  Dissimilation  and  assimilation  are  the  fundamental 

1  See  note,  page  392. 


398  COLLECTED  STUDIES   IN    IMMUNITY 

phenomena  of  metabolism,  while  they  are  also  at  the  same  time  the 
two  phases  of  the  vital  process. 

"  As  a  result  of  a  large  number  of  facts  we  have,  as  is  well  knowny 
arrived  at  the  conclusion,  confirmed  chiefly  by  Pfluger,  that  the  mid- 
point of  metabolism  is  represented  by  complicated  combinations  of 
egg  albumin  called  by  Pfluger  living  albumin.  Such  combinations 
are  exceedingly  labile,  decomposing  to  a  certain  extent  sponta- 
neously, and  to  a  greater  degree  in  response  to  stimuli  In  these 
combinations  we  are  dealing  with  chemical  substances  whose  mole- 
cules, just  because  of  this  easy  decomposition,  disclose  a  chemical 
constitution  quite  different  from  the  lifeless  albuminous  bodies  which 
we  know.  I  have  therefore  proposed  to  replace  the  name  'living 
albumin  molecule'  by  the  term  'biogen  molecule.'  The  decomposi- 
tion and  production  of  the  biogens  is  therefore  the  corner-stone  of  the 
vital  process  in  every  living  cell.  The  substances  given  off  by  the 
cell  are  derived  from  the  decomposition  of  the  biogens;  the  material 
for  the  formation  of  new  biogen  molecules  is  furnished  by  the  food 
taken  up  and  transformed  by  the  cell.  I  have,  however,  called 
attention  to  the  fact  that  this  view  needs  to  be  extended  in  one 
direction  (Allg.  Physiologic,  Jena,  1897).  A  number  of  facts  indi- 
cate that  the  decomposition  of  the  biogen  molecule  is  not  complete 
and  that  all  of  the  atomic  groups  thus  arising  are  not  given  off  by 
the  cell." 

In  view  of  these  explanations  Verworn  assumes  that  in  the  de- 
composition of  the  biogens  a  residue  is  always  left  which  again 
takes  up  lood  substances  and  so  regenerates  the  biogen  molecule. 
It  seems  to  have  entirely  escaped  Verworn  that  I  had  expressed 
entirely  analogous  views  in  much  greater  detail  twelve  years  pre- 
viously ("  Uber  den  Sauerstoffbediirfniss  des  Organismus,"  Berlin, 
1885).  I  assumed  that  the  specific  function  of  the  cell  is  depen- 
dent on  a  central  group  in  the  living  protoplasm,  of  peculiar 
structure;  furthermore,  that  atoms  and  atomic  groups  are  attached 
to  this  central  group  as  side-chains.  These  side-chains  are  of  subordi- 
nate importance  for  the  specific  cell  function,  but  not  so  for  the  life  itself. 
I  also  said  that  everything  indicated  that  it  was  just  through  these 
indifferent  side-chains  that  physiological  combustion  was  effected 
for  one  portion  of  these  side-chains  effects  combustion  by  giving 
off  oxygen,  the  other  portion  being  thus  consumed.  On  page  11  of 
this  monograph  I  expressed  myself  as  follows:  "The  question  as 
to  the  manner  in  which  the  side-chains  constantly  being  consumed 


THE  RECEPTOR  APPARATUS  OF  THE  RED  BLOOD-CELLS.  399- 

are  regenerated  must,  of  course,  excite  the  greatest  interest.  It 
can  be  conceived  that  ceitain  portions  of  the  functional  central 
group  [Leistungskern]  can  fix  combustible  molecular  groups,  and  that 
these  groups  are  thus  rendered  more  susceptible  to  complete  com- 
bustion." 

It  is  at  once  clear  that  these  fixing  portions,  which  I  now  term 
receptors,  correspond  exactly  in  their  nature  to  the  biogen  residues 
of  Verworn. 

Probably  no  one  who  has  seriously  studied  these  questions  will 
question  the  importance  of  these  deductions.  In  spite,  however, 
of  the  decades  which  have  elapsed  since  Pfliiger's  publication  we  have 
not  advanced  one  step  in  our  experimental  knowledge  of  this  sub- 
ject, a  fact  which  is  due  to  the  endless  difficulties  occasioned  by  the 
nature  and  instability  of  the  living  material.  I  hope  that  my  theory 
is  destined  finally  to  bridge  this  wide  gap.  The  knowledge  that  the 
numerous  antibodies  are  nothing  more  than  thrust-off  receptors  of 
the  cell  should  make  it  possible  to  get  at  the  nature  of  assimilating 
processes.  By  means  of  immunization  we  can  compel  the  thrusting- 
off  of  certain  particular  receptors  which  then  collect  in  the  serum. 
Free  from  the  disturbing  connection  with  the  protoplasm,  they  no 
longer  offer  any  difficulties  for  biochemical  investigations.  Viewed 
in  this  light,  I  believe  that  the  facts  which  I  have  determined  con- 
cerning the  action  of  uniceptors  and  amboceptors  constitute  a  new 
step  toward  a  true  conception  of  the  vital  processes. 

It  can  hardly  be  doubted  that  the  red  blood-cells,  owing  to  their 
relatively  simple  structure  and  the  ease  with  which  they  can  be 
manipulated,  are  better  adapted  for  these  purposes  than  other  cellular 
elements.  I  also  believe  that  clinical  investigations  are  destined  to 
play  a  leading  role  in  the  solution  of  these  problems,  simply  because 
the  various  types  of  disease  offer  a  much  greater  variation  in  the  vital 
conditions  than  we  can  attain  by  means  of  experiments.  Even 
aside  from  the  gain  to  pure  biological  science,  clinical  medicine  should 
derive  the  greatest  advantage  from  such  studies,  for,  as  already  men- 
tioned, they  deal  with  the  true  conception  of  the  pathology  of  the 
red  blood-cells. 

In  order  somewhat  to  facilitate  such  a  study  it  may  perhaps  be 
well  to  give  a  brief  sketch  of  the  facts  which  in  conjunction  with 
my  colleague,  Dr.  Morgenroth,  I  have  discovered  regarding  the 
physiology  of  the  receptors. 

Considering  the  large  number  of  receptors  which  each  species 


400  COLLECTED  STUDIES  JN   IMMUNITY. 

of  blood-cell  possesses,  it  is  not  surprising  that  certain  types  are 
common  to  the  majority  if  not  to  all  the  vertebrate  species.  In  this 
connection  I  shall  only  point  out  the  fact  that  receptors  for  ricin, 
abrin,  ichthyotoxin  (which  injure  a  large  number  of  different  erythro- 
cytes)  are  widely  distributed  in  the  animal  kingdom.  Side  by  side 
with  such  generally  distributed  groups,  however,  there  are  types 
which  are  limited  to  a  comparatively  small  group  of  animal  species. 
Thus  by  means  of  cross  immunization  we  have  demonstrated  that  the 
blood-cells  of  goat  and  sheep  possess  several  special  receptors  in 
common.  This  was  shown  by  the  fact  that  the  isolysins  obtained 
by  injecting  goats  with  goat  blood  usually  effected  solution  of  sheep 
blood-cells,  although  to  a  less  degree.  In  making  the  counter  ex- 
periments, immunizing  goats  with  sheep  blood-cells,  we  obtained 
in  addition  to  sheep  lysin  the  isolysin  acting  on  goats. 

Besides  this  there  are  groups  of  receptors  which  are  specific  for 
each  animal  species.  This  is  best  shown  by  the  normal  course  of 
the  Belfanti-Bordet  experiments.  In  these  as  a  rule  only  specific 
hsemolysins  are  formed,  i.e.,  hsemolysins  directed  against  the  erythro- 
cytes  exciting  the  immunization.1 

Such  variations  in  the  zoological  distribution  of  certain  recep- 
tors (also  of  the  complements,  etc.)  is  readily  explained  by  the  very 
natural  assumption  that  the  metabolic  processes,  whose  indicator 
the  receptors  really  are,  show  corresponding  variations.  It  is  just 
as  little  to  be  doubted  that  certain  assimilative  processes  are  specific 
for  only  one  species  of  animal  as  that  others  occur  in  exactly  the 
same  manner  in  man  and  in  the  frog. 

It  is  also  of  considerable  importance  that  in  any  given  animal 
species  a  considerable  individual  variation  of  the  receptors  may  occur, 
a  fact  first  observed  in  experiments  with  crotin  on  rabbits.  The 
strongest  confirmation  of  this  point  is  the  result  of  our  experiments 
on  goat  isolysins.  As  already  stated,  out  of  the  goats  we  used  there 
were  always  only  a  few  which  reacted  to  one  of  the  thirteen  different 
isolysins. 

Through  the  opportunity  so  offered  we  convinced  ourselves  of 
another  important  fact,  namely,  that  the  susceptibility  of  a  given 
individual  can  change  in  a  comparatively  short  time.  We  found 
that  a  goat  which  reacted  to  a  certain  isolysin  became  unsuscep- 


1  We  have  obtained  entirely  analogous  results  also  with  other  constituents 
of  blood  serum,  e.g.,  with  complements. 


THE  RECEPTOR  APPARATUS  OF    THE  RED  BLOOD-CELLS.   401 

tible  after  several  weeks,  and  further  that  in  this  case  there  had 
been  a  disappearance  of  the  special  receptors  previously  demon- 
strated as  present.  We  have  also  encountered  the  reverse  of  this, 
namely,  the  appearance  of  receptors  previously  absent. 

Evidently  this  coming  and  going  of  certain  receptors  reflects 
internal  metabolic  processes  which  may  be  dependent  on  a  large 
number  of  external  or  internal  factors.  In  this  connection  a  fact 
observed  by  Kossel  is  especially  interesting.  This  observer  found 
that  during  the  course  of  immunization  with  eel  blood  the  blood- 
cells  of  rabbits  acquire  a  high  degree  of  resistance  against  the  poison, 
a  fact  which  we  should  perhaps  ascribe  to  a  lack  of  receptors.  In 
this  case  we  are  dealing  with  something  which  is  specific  for  the 
immunization  with  eel  blood,  for  we  could  not  obtain  these  results 
with  two  other  blood  poisons,  crotin  and  tetanolysin. 

To  a  certain  extent  the  experiments  of  Kossel,  Gley,  and  Tschis- 
towitsch  furnish  a  clue  to  the  mechanism  of  these  phenomena.  They 
show  that  the  first  phase  of  immunization  is  that  of  antitoxin 
formation,  and  that  the  unsusceptibility  of  the  red  blood-cells  is 
not  developed  until  later. 

The  way  in  which  blood-cells  which  have  previously  been  sus- 
ceptible to  a  certain  poison  become  unsusceptible  to  this  can  very 
readily  be  explained.  We  have  seen  that  those  blood-cells,  which 
are  susceptible  to  the  action  of  a  poison  (e.g.,  eel  blood)  possess 
appropriate  receptors.  Under  physiological  conditions  the  office  of 
these  is  to  anchor  a  certain  particular  product  of  metabolism,  x.  If 
now  through  treatment  with  the  poison  the  specific  antitoxin  is 
produced,  it  is  clear  that  this  antitoxin  when  present  in  the  circu- 
lation is  able  to  anchor  not  only  the  poison  but  also  the  normal  meta- 
bolic product,  x,  thus  preventing  the  latter  from  combining  with  the 
erythrocytes.  Since  this,  however,  renders  the  corresponding  recep- 
tors permanently  useless,  the  possibility  of  their  disappearance  is  at 
once  given— after  the  manner  of  atrophy  through  disuse.  This  will 
occur  most  readily  in  those  cases  in  which  the  substance  x  can 
readily  be  spared  by  the  cell,  i.e.,  cases  in  which  (as  in  sugar) 
the  substance  can  be  replaced  by  some  other  kind  of  material 
(e.g.,  fat). 

A  disappearance  of  the  receptors  can,  however,  occur  without 
the  development  of  such  a  deflecting  antibody,  as  is  shown  by  the 
isolysin  experiments.  The  most  natural  conclusion  is  that  the  lack 
of  receptors  in  this  case  is  produced  by  an  inconstant,  perhaps  only 


402  COLLECTED  STUDIES   IN   IMMUNITY 

a  temporary,  metabolic  product.  Perhaps  this  can  be  brought  into 
connection  with  the  interesting  observation  of  Gley  that  the  blood- 
cells  of  new-born  rabbits  are  highly  resistant  against  eel  poison, 
acquiring  the  normal  high  susceptibility  only  in  the  course  of 
weeks. 

Be  this  as  it  may,  everything  indicates  that  there  is  an  organic 
harmonious  connection  between  the  metabolism  of  any  given  period 
and  the  nature  of  the  receptors  present.  This  connection  depends  on 
the  fact  that  substances  with  haptophore  groups  exert  a  stimulus 
on  the  protoplasm  which  excites  the  production  of  the  receptors  in 
question. 

In  conclusion  I  wish  to  point  out  that  many  facts  indicate  that 
the  species  of  receptors  found  in  the  erythrocytes  may  also  be  present 
in  the  cells  of  other  organs.  Thus,  mentioning  only  one  example, 
tetanolysin  is  anchored  not  only  by  the  erythrocytes,  but  also  by 
the  brain  and  other  organs.  This  phenomenon  also  shows  itself  in 
the  immunizing  test.  Von  Dungern,  for  example,  found  that  serum 
of  rabbits  which  had  been  treated  with  tracheal  epithelium  of  oxen 
exerted  a  marked  hsemolytic  action  on  ox  blood  in  addition  to  its 
injurious  action  on  epithelium.  Metchnikoff's  objection  that  this 
was  due  to  an  error  in  technique  (the  injection  of  admixed  blood- 
cells)  was  controverted  by  von  Dungern,  who  showed  that  injections 
of  cow  milk,  a  material  absolutely  free  from  blood-cells,  produced 
the  same  haemolysins.  It  follows  that  certain  receptors  must  be 
common  to  the  red  blood-cells  and  the  epithelial  tissue  or  the  milk 
derived  from  this. 

The  wide  distribution  of  a  particular  combining  group  harmonizes 
very  well  with  the  assumption  discussed  above  concerning  the  func- 
tions of  the  receptor  apparatus  of  the  red  blood-cells. 

According  to  Miescher's  comparison  the  red  blood-cells  serve  as 
a  sort  of  bank  of  deposit  where  the  metabolic  products  in  excess  at 
any  given  time  may  be  stored  temporarily.  In  this  case  the  sub- 
stances will  be  yielded  up  only  to  organs  possessing  suitable  receptors. 
This  process  will  be  all  the  more  complete  if  the  affinity  of  the  tissue 
receptors  is  greater  than  that  of  the  blood  receptors.  There  are 
many  reasons  for  believing  that  the  affinity  of  the  tissue  receptors 
is  not  constant,  and  that  it  can  be  considerably  increased  through 
certain  stimuli  (assimilative  stimuli).  It  is  obvious  that  hunger,  if 
we  may  apply  the  term  to  purely  cellular  processes,  must  constitute 
one  of  the  most  important  assimilative  stimuli.  This  functional  in- 


THE   RECEPTOR   APPARATUS  OF    THE   RED   BLOOD-CELLS.  403 

crease  of  affinity  would  constitute  a  wonderful  illustration  ot  how 
well  the  process  of  assimilation  is  adapted  to  its  purpose. 

NOTE.— Subsequent  addition  to  page  400: 

Calmette  also  has  recently  reported  (Compt.  rend,  de  1' Academic  des  sciences-, 
T.  134,  No.  24,  1902)  that  the  blood-cells  of  animals  highly  immunized  with 
cobra  poison  preserve  their  sensitiveness  completely  against  the  haemolysin 
of  the  cobra  poison,  In  a  goat  highly  immunized  with  ricin,  Jacoby  (Hof- 
meister's  Beitrage  z.  chem  Physiologic  und  Pathologie,  Bd.  II,  1902)  was 
unable  to  discover  any  increased  resistance  of  the  red  blood-cells  against  the 
action  of  the  ncin. 


XXXIV.  THE  RELATIONS  EXISTING  BETWEEN   CHEM- 
ICAL   CONSTITUTION,  DISTRIBUTION,    AND 
PHARMACOLOGICAL  ACTION.1 

(An  Address  delivered  in  the  "Verein  fiir  innere  Medicin,"  Dec.  12,  1898.) 
By  Professor  Dr.  P    EHRLICH. 

UNTIL  recent  years  the  relations  between  chemistry  and  medicine 
were  in  general  confined  to  purely  scientific  questions.  In  the  last 
decade,  however,  a  change  has  taken  place,  such  as  has  rarely  been 
seen  in  the  history  of  medicine.  One  is  justified  in  saying  that  at 
the  present  time  the  chemical  view  constitutes  the  axis  about  which 
the  most  important  views  in  medicine  turn,  and  that  the  two  poles 
are  the  synthetic  construction  of  new  therapeutic  agents  on  the 
one  hand,  and  the  discovery  of  specific  therapeutic  products  of 
living  cells  on  the  other.  The  contrast  between  these  two  methods 
is  very  pronounced.  In  the  first  case,  one  makes  use  of  the  retort 
and  simple,  definite  reactions;  in  the  other,  of  the  mysterious  powers 
of  living  nature  so  infinitely  well  suited  to  their  purpose.  A  greater 
contrast  cannot  be  imagined  than  that  existing  between  the  modern 
medicaments,  whose  constitution  is  known  down  to  the  finest  details, 
and  diphtheria  antitoxin,  which  we  know  only  through  its  specific 
action  and  about  whose  chemical  constitution  we  know  absolutely 
nothing.  Thus  far  the  genius  of  the  most  eminent  chemists  has  not 
availed  to  produce  these  bodies  in  a  pure  form  and  get  an  insight 
into  their  chemical  nature.  All  that  this  endless  study  has  brought 
forth  is  the  conviction  that  we  are  dealing  with  atomic  groups  of 
the  utmost  complexity,  which  for  the  present  are  entirely  beyond 
our  chemical  researches  and  which,  so  far  as  we  can  see,  will  long 
remain  so. 

1  Reprint  from  the  v.  Leyden  Festschrift,  Vol.  I. 

404 


CHEMICAL  CONSTITUTION    AND   PHARMACOLOGICAL  ACTION.     405 

As  a  result  of  this  and  other  considerations  the  view  has  become 
prevalent  that  the  chemo-therapeutic  and  the  bio-therapeutic  ten- 
dencies are  absolutely  different  from  each  other.  As  late  as  two 
years  ago  a  certain  high  authority  said  that  the  antitoxins  act  after 
the  manner  of  specific  forces  (in  a  physical  sense).  If  this  theory 
of  "forces"  were  to  be  upheld  every  possibility  of  bridging  the  con- 
tradictions would  be  completely  lost,  for  then  every  tertium  compara- 
tionis  would  be  lacking. 

If  instead  of  this  we  assume  that  both  kinds  of  substances  exert 
their  power  by  purely  chemical  means,  we  shall  find  that  certain 
questions  arise  which  are  of  great  significance  for  the  further  develop- 
ment of  therapeutics.  Convinced  that  this  is  correct  I  have  busied 
myself  during  the  past  ten  years  with  attempts  to  prove  the  chemical 
theory  of  toxins  and  antitoxins  experimentally.  I  believe  I  am 
justified  in  claiming  that  I  have  caused  the  chemical  conception  to 
be  accepted  among  ever-widening  circles.  This  1  have  accomplished : 

1.  By  the  introduction  of  the  test-tube  experiments. 

2.  By  systematic  investigations  concerning  the  mutual  satisfying 

affinities. 

3.  By  the  demonstration  of  toxoids  and  their  various  modifications. 


I. 

If  then  the  medicaments  of  known  constitution  and  the  biothera- 
peutic  products,  both  act  only  in  a  chemical  manner,  i.e.,  if  both 
effect  the  organism  chemically,  the  first  problem  to  be  solved  is  to 
determine  on  what  factor  the  very  dissimilar  action  of  these  two 
classes  of  bodies  depends.  It  will  be  well  to  begin  with  the  simplest 
condition,  and  to  study  first  the  mode  of  action  of  bodies  whose 
chemical  constitution  is  well  known. 

It  is  particularly  desirable  to  gain  an  insight  into  the  relations  exist- 
ing between  chemical  constitution  and  pharmacological  action.  Dur- 
ing the  last  few  decades  these  have  come  to  play  an  important  role  in 
the  modern  synthetic  tendencies.  The  history  of  this  tendency  is 
comparatively  recent,  dating  from  the  year  1859  when  Stahlschmidt 
demonstrated  that  strychnine  loses  its  tetanizing  action  when  a 
methyl  group  is  introduced,  being  transformed  into  a  curare-like 
poison.  In  view  of  the  fact  that  this  methylation  forms  an  ammo- 
nium base,  Fraser  and  Braun  studied  a  number  of  other  ammonium 
bases  derived  from  various  alkaloids  and  found  that  all  of  these  bodies 


406  COLLECTED   STUDIES  IN    IMMUNITY. 

possessed  a  curare-like  action.  Since  that  time  a  large  number  of 
ammonium  bases  derived  from  the  most  varied  alkaloids  have  been 
investigated,  most  all  of  which  showed  the  same  action.  The  final 
step  was  achieved  only  recently  when  Bohm  showed  that  curarin  is 
itself  an  ammonium  base.  He  found  that  the  curares  contain  a 
tertiary  alkaloid,  curin,  which  is  of  slight  toxicity.  If  this  curin 
wras  subjected  to  methylation  an  ammonium  base  was  formed  which 
corresponded  completely  in  properties  and  actions  with  the  natural 
curarin,  but  was  about  260  times  as  toxic  as  the  original  substance. 
Since  this  time  these  questions  have  been  studied  on  many  different 
combinations  by  a  large  number  of  investigators,  among  whom 
may  be  mentioned  Nencki,  Jaff^,  Filehne,  Mering,  Brunton,  Brieger, 
Gibbs,  and  Aronson.  I  cannot,  however,  go  into  details  and  must 
confine  myself  to  giving  a  short  epitome  of  what  has  been  done  in 
the  development  of  synthetic  remedies. 

First  in  importance  are  the  artificial  antipyretics,  of  which  the 
main  types  are  the  antipyrin  series  and  the  phenacetin  series.  The 
history  of  the  origin  of  these  two  groups  is  absolutely  unlike.  In 
one  case  the  starting-point  was  the  fact  that  quinine  contains  a 
hydrated  chinolin  derivative;  by  means  of  simpler  combinations  it 
was  attempted  to  obtain  the  same  end.  Finally,  after  chinolin, 
kairin  and  thallin  had  proved  of  such  little  value,  antipyrin  was 
obtained  and  found  most  useful.  The  second  group,  which  includes 
phenacetin  and  its  numerous  relatives,  owes  its  discovery  not  to 
theoretical  speculations  but  to  a  coincidence,  the  result  of  an  error. 

Of  the  other  therapeutic  agents  the  discovery  of  the  hypnotic 
action  of  sulfonal  by  Baumann  has  proven  of  great  practical  and 
theoretical  significance.  The  same  holds  true  of  the  production  of 
the  new  anaesthetics  (orthoform  and  eucain),  which  was  closely  con- 
nected with  the  discovery  of  the  constitution  of  cocaine.  In  recent 
years  efforts  are  constantly  being  made  to  do  away  with  the  by- 
effects  possessed  by  certain  remedies,  such  as  guaiacol  and  formal- 
dehyd.  These  efforts,  first  undertaken  by  Nencki,  seek  by  means 
of  suitable  combinations  and  cleavages  to  give  rise  to  a  gradual 
liberation  of  the  active  component.  While  of  great  practical  value 
they  have  but  little  interest  in  the  question  concerning  the  connection 
between  constitution  and  action. 

When  now  we  come  to  inquire  what  conclusions  we  can  draw 
from  the  study  of  the  large  number  of  therapeutic  agents,  which  now 
embrace  many  hundreds  of  different  remedies,  conclusions  which 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.     407 

will   apply  to  the  study  of  the  relation  between  constitution  and 
action,  we  find  that  the  results  are  still  very  meagre. 
In  the  main  they  are  as  follows : 

1.  The  discovery  that  the  antipyretic  action  of  the  anilin  and 
amidophenol    derivatives    (phenacetin)    is   proportional,  within   cer- 
tain limits,  to  the  amount  of  p-amidophenol  split  off  in  the  organism 
(Hinsberg).     Hence   all  such   combinations   in   which,   through  im- 
proper substitution  of  the  amido  group  or  of  the  main  group  (p- 
amidoacetophenon,  NH2-C6H4-CO-CH3)?  the  liberation  of  p-amido- 
phenol is  prevented  cannot  be  used  as  antipyretics. 

2.  The  discovery  by  Kendrick,  Dewar,  Filehne,  that  in  the  pyri- 
din  series  the  hydrated  products  act  more  strongly  than  the  parent 
substance.      Thus  piperidin,  C5H10NH,  is  a  much  stronger  poison 
than  pyridin,  C5H5X.     In   this  the  transformation  of  the  tertiary 
nitrogen  atom  in  the  imin  group  plays  a  certain  role,  as  is  shown 
especially  by  the  observations  of  Filehne  on  the  tetra-hydro-chinolm 
series.     According  to  these  the  replacement  of  the  imid's  hydrogen 
atom  by  alcohol  radicals  reduces  the  irritant  action. 

3.  The  demonstration  that  the  antipyretic  power  of  antipyretics 
is  destroyed  by  the  introduction  of  salt-forming  acid  radicals,  such 
as    SO3H,    CO2H    (Ehrlich,    Aronson,    Nencki,    Penzoldt).      Hence 
so     far     as     this     action     is      concerned     acetanilido-acetic     acid, 
C6H5X(COCH3)CH2CO2H,  is  inert.      So  also  are  acetanilin  sulfonic 
acid,  C6H5  •  XH  •  CO  •  CH2SO3H,  the  carbonic  and  sulfonic   acids  of 
phenacetin,    and    the     ethoxy-phenylglycin    which    is    similar    to 
phenacetin. 


C6H4 


/OC2H5 
\XH-CH2.CO2H. 


4.  The  demonstration  by  Filehne,  Einhorn,  Ehrlich,  and  Poulson, 
of  the  ansesthesiophore   character  of  the  benzoyl  radical.     Homo- 
logues  of  cocaine,  such  as  are  obtained  when  other  acid  radicals, 
such  as  succinic  acid,  phenylacetic  acid;  cinnamic  acid,  are  intro- 
duced into  the  ecgoninmethylester,  lack  these  anaesthetic  properties. 
This  discovery  resulted  in  the  production  of  new  potent  anaesthetics 
containing  the  benzoyl  group  as  the  active  agent,  e.g.  eucain  (Merling) 
and  orthoform  and  nirvanin  (Einhorn). 

5.  The  function  of  the  ethyl  group.     This  has  been  brought  out 
very  clearly  by  Baumann's  discovery  that  the  hypnotic  action  of 
certain  disulfons  is  due  exclusively  to  the  presence  of  ethyl   groups 


408  COLLECTED  STUDIES  IN   IMMUNITY. 

and  that  it  increases  with  the  number  of  these  groups:  thus  sulfonal, 
(CH3)2.C-(S02C2H5)2,  and  trional,  CH3C2H5-C- (S02C2H5)2.  Of 
the  other  hypnotics  which  owe  their  action  in  part  to  the  ethyl  group 
I  may  mention  amylenhydrate,  C(CH3)2(C2H5) -OH,  and  ethyl  ur- 
ethan,  NH2  •  CO  •  OC2H5.  The  influence  of  the  ethyl  radical  is  further- 
more clearly  shown  in  another  series  of  combinations.  In  an  artificial 
sweetening  substance,  dulcin,  which  is  about  two  hundred  times  sweeter 
than  sugar,  this  influence  is  very  evident.  This  substance  is  phenyl 
urea  ethoxylated  in  the  para  position,  C2H50-C6H4-NH  CO-NH2. 
Since  neither  simple  phenyl  urea  nor  the  methoxy  combination, 
CH3-0-C6H4-NH-CO-NH2,  analogous  to  dulcin,  possesses  any  sweet 
taste  whatsoever,  we  are  forced  to  conclude  that  this  is  due  to  a  function 
of  the  ethyl  radical.  Of  the  "remedies  containing  the  ethyl  radical 
there  may  still  be  mentioned  phenacetin,  C2H5  •  O  •  CoH4  •  NH  •  CO  •  CH3, 
and  two  anaesthetics,  holocain,  C2H5-O-C6H4-NH-C(CH3): 
N  •  CeH4  •  OC2H5,  and  acoin,  all  three  of  which  are  derived  from 
phenetidin.  It  is  significant  that  of  the  entire  series  of  alcohols 
only  ethyl  alcohol  has  become  established  as  a  beverage,  and  that 
since  the  earliest  time  attention  was  directed  to  producing  it  as  pure 
as  possible,  i.e.,  to  free  it  from  higher  and  lower  relatives.  In  all 
of  these  examples  we  are  dealing  with  an  influence  on  the  nervous 
system,  the  central  system  (sulfonal  ethylurethan,  amylen  hydrate, 
alcohol),  as  well  as  the  peripheral  endings  (dulcin,  anaesthetics). 
Hence  we  shall  probably  not  err  if  we  assume  that  the  ethyl  group 
possesses  a  certain  relation  to  the  nervous  system.  In  this  con- 
nection an  observation  which  I  made  in  conjunction  with  Dr.  Michaelis 
may  perhaps  be  of  some  significance.  We  were  studying  a  blue-green 
azo  dye  which  is  formed  by  the  combination  of  diazotated  diethyl- 
saffranin  and  dimethylanilin,  and  which  therefore  is  expresed  by 
the  formula 


— N  =  N 


\ 

N(CH3)2 


It  was  found  that  this  substance  has  the  power,  somewhat  like 
methylene  blue,  to  stain  the  nerve  endings  of  living  (?)  tissue  organs, 


CHEMICAL  CONSTITUTION   AND   PHARMACOLOGICAL  ACTION      409 

whereas  the  corresponding  dyes  derived  from  saffranin,  tolusaffranin, 
and  dimethyl-saffranin  do  not  possess  this  property.  Some  time  after 
this  we  received  a  second  dyestuff,  of  unknown  constitution,  which 
possessed  the  same  neurotropic  properties,  and  we  therefore  at  once 
assumed  that  this  body  also  contained  a  diethylanilin  radical.  On 
inquiry  of  the  manufacturer  we  found  our  conjecture  verified.  This 
staining  experiment  may  perhaps  afford  valuable  confirmation  of  the 
view  expressed  above  concerning  the  function  of  the  ethyl  radical. 

This  synopsis  will  show  that  our  actual  knowledge  concerning  the 
relation  between  constitution  and  action  is  still  in  its  very  infancy. 
Hence  the  expectation  to  be  able  to  construct  new  remedies  of  pre- 
determined action  on  the  basis  of  theoretical  conceptions  will  prob- 
ably have  to  be  deferred  for  a  long  time.  To  the  initiate  the  lack 
of  sufficient  positive  knowledge  is  revealed  by  the  inactivity  which 
now  characterizes  a  field  once  entered  upon  with  so  much  promise. 
The  innumerable  remedies  which  have  overwhelmed  medicine  in  the 
past  few  years,  of  which  only  a  few  are  of  any  value,  and  thus  denote 
any  real  progress,  have  sufficed  speedily  to  allay  the  original  enthu- 
siam.  A  feeling  of  indifference  has  thus  been  engendered  which  is 
constantly  being  increased  by  the  advertisements  which  are  daily 
becoming  more  and  more  evident.  Aside  from  these  evils,  however, 
this  line  of  study  is  at  present  suffering  especially  from  two  other 
evils : 

1.  The  habit,  when  a  remedy  has  been  partly  accepted,  of  imme- 
diately following  it  with  a  dozen  rivals  of  similar  composition. 

2.  The   exclusive    preference   given    to    remedies    acting    purely 
symptomatically,  which  are  not  true  curative  agents. 

A  change  for  the  better  will  only  then  occur  if  pure  biological 
points  of  view  are  adopted,  i.e.,  if  the  initiative  is  transferred  from 
the  chemical  to  the  biological  laboratory.  As  physicians  we  must 
stop  remaining  content  with  the  auxiliary  role  of  counsel  in  these 
important  questions.  In  this  subject,  our  very  own  since  time 
immemorial,  we  must  insist  on  taking  first  place.  Just  now  it  is 
essential  that  we  gain  more  general,  biological  conceptions,  and  it 
is  therefore  every  one's  duty  to  contribute  his  mite  to  the  develop- 
ment of  this  therapy. 


410  COLLECTED  STUDIES  IN   IMMUNITY. 


II. 

One  of  the  main  causes  which  has  made  an  insight  into  the  rela- 
tion between  constitution  and  action  so  difficult  to  obtain  is  to  be 
found  in  the  fact  that  these  relations  were  considered  to  be  much 
simpler  than  they  really  are,  and  in  the  further  fact  that  purely 
chemical  conceptions  were  applied  arbitrarily  to  biological  processes. 
In  pure  chemistry  there  is  an  abundance  of  material  for  observing 
the  relations  between  physical  properties  and  chemical  constitu- 
tion. In  such  a  study  it  is  first  necessary  to  determine  which  proper- 
ties, to  follow  Ostwald's  terminology,  are  " additive"  and  which 
"constitutive  "  by  nature. 

The  question  arises  what  are  the  essential  properties  which  are 
;still  found  in  the  combinations.  Evidently  they  are  such  as  per- 
tain to  the  substance  of  the  elements  and  are  independent  of  the 
arrangement  of  these.  These  properties  accompany  the  elements  in 
their  combinations,  assuming  therein  values  which  represent  the  sum 
of  the  values  of  the  elements.  In  other  words  these  are  "additive" 
properties. 

Real  additive  properties  are  not  known  apart  from  mass.  The 
neaiest  approach  to  them  are  perhaps  the  specific  heat  of  solid  com- 
binations, and  in  a  less  degree  the  refraction  of  organic  substances  and 
their  property  to  occupy  space.  In  these,  however,  another  factor 
becomes  evident,  namely,  the  arrangement  of  the  elements  in  their 
combinations.  This  factor  is  of  paramount  importance  in  deter- 
mining such  properties  as  color,  boiling-  and  melting-point,  form  of 
crystals,  etc.  The  properties  which  are  under  the  mutual  control 
of  the  nature  of  the  elements  and  their  arrangement  are  called 
"constitutive  "  properties.  The  extreme  in  this  direction  is  made 
up  of  those  properties  which  are  no  longer  in  any  way  dependent 
on  the  nature  of  the  substances  but  only  on  their  arrangement ; 
these  are  called  "  colligative  "  properties. 

To  which  group,  then,  do  the  properties  of  affinity,  i.e.,  the  power 
of  elements  to  effect  chemical  reactions,  belong?  Evidently  to  the 
constitutive,  for  daily  experience  teaches  us  that  the  nature  as  well 
as  the  arrangement  of  the  elements  is  a  factor.  Acetic  acid,  lactic 
acid,  and  glucose  contain  the  same  elements  in  the  same  propor- 
tions by  weight,  yet  they  manifest  entirely  different  reacting  capaci- 
ties. Butyric  acid  and  acetic  ester  are  not  only  of  the  same  con- 


CHEMICAL    CONSTITUTION  AND   PHARMACOLOGICAL  ACTION.    4U 

stitution  but  have  the  same  molecular  weight,  yet  their  affinities 
are  different.1 

There  is  probably  no  doubt  that  those  properties  of  organic  sub- 
stances which  interest  us  as  therapeutists  are  constitutive  in  nature. 

R.  Meyer  has  published  a  most  interesting  article  on  certain  re- 
lations between  fluorescence  and  chemical  constitution.  In  this  he 
calls  attention  to  the  fact  that  the  relations  between  the  color  of 
chemical  combinations  and  their  constitution  have  not  up  to  the 
present  time  been  studied  with  the  exactness  with  which  charac- 
teristics less  apparent  have  been  examined,  such  as  rotation  and 
the  refractive  index.  The  reason  for  this  is  that  the  refractive  index 
of  a  body  is  a  definite  number,  the  specific  rotation  an  angle  whose 
size  can  be  exactly  determined,  whereas  color  is  more  qualitative 
in  character,  and,  strictly  speaking,  is  not  a  physical  but  a  physio- 
logical characteristic.  A  body  which  possesses  strong  ultraviolet 
absorption  bands  is  colorless  to  our  eyes,  yet  it  may  appear  colored 
to  a  visual  organ  differently  constituted  than  ours.  We  see,  therefore, 
that  even  in  so  conspicuous  a  property  as  color  the  physiological 
factor  interferes  with  our  gaining  a  clear  insight  into  the  relations 
existing  between  constitution  and  action.  It  will  at  once  be  con- 
ceded that  this  is  true  to  a  still  greater  degree  in  the  complex  processes 
which  underlie  pharmacological  action. 

But  it  is  just  because  of  this  intermediate  position  that  the  chem- 
istry of  dyestuffs  affords  so  good  a  point  of  vantage  for  our  con- 
sideration. I  may  therefore  perhaps  be  permitted  to  briefly  outline 
what  has  thus  far  been  learned  concerning  the  relations  between 
color  and  constitution,  especially  in  view  of  the  fact  that  I  shall 
frequently  have  to  touch  on  the  biology  of  dyes  in  the  succeeding 
chapters. 

In  1868  C.  Graebe  and  C.  Liebermann  demonstrated  that  color 
was  in  some  way  associated  with  a  certain  denser  combination  of  the 
atoms.  If  this  is  overcome  by  the  addition  of  hydrogen  the  color 
will  disappear, 'the  dye  passing  into  the  "leuco"  combination  (thus 
indigo  into  indigo  white),  out  of  which  it  can  again  be  produced  by 
oxidation. 

A  great  advance  was  then  made  by  (X  N.  Witt,  who  showed  that 
the  color  properties  of  a  dyestuff  are  due  to  the  presence  of  a  certain 
unsaturated  group  of  atoms  which  he  terms  the  color-producing  or 

1  Ostwald,  Grundriss  der  allgemeinen  Chemie. 


412  COLLECTED  STUDIES  IN   IMMUNITY 

"chromophore  "  group.  Concerning  the  deatils  of  the  various  types 
of  chromophores  I  refer  the  reader  to  the  admirable  work  of  Nietzki. 
I  may,  however,  say  here  that,  as  a  rule,  the  action  of  the  chromophore 
groups  as  such  does  not  become  manifest  if  the  group  is  part  of  a 
molecule  very  poor  in  carbon  atoms.  Hence  colored  combinations 
are  rare  in  the  fatty  series;  they  belong  almost  exclusively  to  the 
aromatic  series  (Nietzki).  The  presence  of  a  chromophore  group 
does  not,  however,  by  itself  suffice  to  produce  true  dyes.  Thus 
azobenzol,  which  possesses  the  chromophore  azo  group,  N=N,  is 
no  dye,  because  it  possesses  no  affinity  for  tissues.  For  this  reason 
Nietzki  terms  azobenzol  a  "chromogen,"  i.e.,  a  combination  which 
becomes  a  true  dye  when  suitable  groups  are  introduced.  Radicals 
which  have  the  power  to  develop  the  nature  of  a  dye  are  called 
"  auxochrome  "  radicals  (Witt).  Thus  far  we  know  but  two,  namely, 
the  OH  group  which  produces  dyes  of  an  acid  character,  and  the 
amido  group  which  produces  basic  dyes.  In  contrast  to  this  it  is 
found  that  other  salt-forming  groups  are  not  auxochromic.  This 
holds  true  not  only  for  acid  complexes,  such  as  the  carboxyl  group 
and  the  radical  of  sulpho  acids,  but  also  for  certain  basic  radicals 
as  NH4,  CH2-NH2,  CH2-N-(CH3)2,  and  O-CH2  N •  (CH3)2. 

From  every  chromogen,  therefore,  two  series  of  dyes  may  be  de- 
rived, acid  and  basic,  each  acid  derivative  having  an  analogous  basic 
one.  Thus 

Acid  Basic 

Oxyazobenzol Amidoazobenzol 

Dioxyazobenzol  (resorcin  yellow).  . Diamidoazobenzol  (chrysoidiny 

Rosolic  acid.     Rosanilin 

Thionol Thionolm 

Aposaffranon Aposaffranin 

If  several  similar  auxochromes  are  introduced  into  a  chromogen 
it  will  be  found  that  up  to  a  certain  point  the  intensity  of  the  shade 
and  the  affinity  for  the  tissues  increases  with  the  number  of  groups  in- 
troduced; thus,  amidoazo benzol — yellow;  diamidoazo benzol — orange;, 
triamidoazobenzol — brown . 

Witt's  observations  extended  only  to  the  question  whether  and 
under  what  conditions  a  body  is  colored,  Nietzki  went  a  step  fur- 
ther and  showed  that  the  simplest  azo  bodies,  as  also  all  the  most 
simply  constituted  dyes,  possess  a  yellow  color.  He  showed  that 
the  tint  deepens  not  only  with  the  increase  in  auxochrome  groups 
just  mentioned,  but  also  with  the  accumulation  ol  carbon  atoms  in. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.    413 

the  molecule.  In  many  cases  the  color  thus  passes  through  red  into 
violet,  in  other  cases  it  passes  into  brown.  Besides  this  the  chemistry 
of  the  rosanilin  dyes  furnishes  many  examples  of  change  in  tint  through 
the  introduction  of  substituting  groups;  thus,  rosanilin — red;  tri- 
methylrosanilin — red  violet;  hexamethylrosanilin — blue  violet;  tri- 
phenylrosanilin — blue. 

I  may  add  that  in  several  cases  these  views  have  been  applied 
also  to  bodies  possessing  physiological  action.  In  cocaine,  for  ex- 
ample, the  ester-like  benzoyl  radical,  (CO-C6H5),  undoubtedly  repre- 
sents the  anesthesiophore  group;  the  tertiary  amin  contained  in  the 
basic  portion  representing  an  analogue  of  the  auxochrome  group, 
and  hence  called  auxotox.  This  is  borne  out  by  the  fact  determined 
by  me  that  cocaine  loses  its  anesthetizing  properties  when  through 
methylation  the  tertiary  amin  is  converted  into  a  quaternary  ammo- 
nium base.  Analogous  to  this  is  the  fact  that  through  complete 
methylation  tertiary  groups  lose  the  property  to  act  as  auxochromes, 
for  the  ammonium  radicals  thus  formed  merely  give  rise  to  an  in- 
creased solubility.  Thus  through  the  introduction  of  a  methyl  group, 
hexamethyl  violet,  which  possesses  three  dimethylamido  radicals, 
passes  over  into  the  soluble  methyl  green,  which  possesses  two  di- 
methylamido groups  and  one  ammonium  group.  Hence  methyl  green 
is  a  triphenyl-methan  dye  which  contains  two  dimethylamido  groups 
as  auxochromes.  In  this  it  is  like  malachite  green,  which  it  therefore 
matches  entirely  in  tint. 

The  third  portion  of  the  cocaine  molecule,  the  carboxylmethyl 
group,  COOCH3,  on  the  other  hand,  is  probably  of  but  little  im- 
portance, as  can  be  seen  from  the  strong  anesthetic  action  of  benzoyl- 
pseudotropein,  which  does  not  possess  this  group. 


III. 

Having  thus  briefly  sketched  some  of  the  more  important  points 
concerning  the  relation  between  chemical  constitution  and  action, 
I  pass  on  the  pharmacological  side  of  the  subject,  in  which,  to  be 
sure,  the  conditions  are  far  more  complex.  It  will  be  well  to  com- 
mence with  a  very  simple  example.  We  know  a  large  number  of 
poisons  which  through  appropriate  substitution  are  practically  de- 
prived of  their  deleterious  action.  As  was  shown  by  Aronson  and 
myself,  this  is  true,  especially  of  the  radicals  of  sulphuric  and  carbonic 
acids.  Independently  of  us,  Xencki  came  to  the  same  conclusion. 


414  COLLECTED  STUDIES  IN   IMMUNITY 

Thus  by  allowing  sulphuric  acid  to  act  on  anilin,  which,  as  is  well  known, 
is  highly  toxic,  the  toxicity  is  completely  destroyed,  for  the  result- 
ing sulfanilic  acid  can  be  taken  in  large  doses  without  injury.  In 
like  manner  the  amidobenzoic  acids  are  non-toxic;  so  also  the  meta- 
and  para-oxybenzoic  acids  derived  from  phenol,  while  the  ortho 
isomer  (salicylic  acid)  still  exhibits  the  familiar  toxic  effects,  although 
they  are  far  less  intense  than  those  of  phenol.  These  surprising 
results  cannot  be  ascribed  to  purely  chemical  effects,  as,  for  example, 
by  assuming  that  the  acid  derivatives  are  more  difficult  to  oxidize  than 
the  original  substance  and  that  they  therefore  do  not  abstract  oxygen 
from  the  tissues.  Certain  observations,  however,  which  I  had  made 
many  years  previously  in  connection  with  vital  staining  furnish  a 
very  simple  explanation.  I  found  that  the  power  to  stain  gray  nerve 
tissue  is  possessed  by  only  a  small  number  of  dyes,  and  especially 
by  certain  basic  dyes  (chrysoidin,  Bismarck  brown,  neutral  red, 
phosphin,  flavanilin,  methylene  blue),  whereas  of  the  acid  dyes,  in 
which  OH  constitutes  the  auxochrome  group,  only  one,  alizarin, 
possesses  this  property.  All  dyes  which  contained  a  sulphuric  acid 
radical  were  absolutely  negative,  and  I  examined  a  very  large  number. 
What  is  especially  significant  is  that  even  neurotropic  stains  lost  this 
property  entirely  if  sulfonic  acids  were  introduced,  a  fact  demonstrated 
in  the  flavanilin  sulfonic  acids,  the  alizarin  sulfonic  acids,  and  the 
sulfonic  acids  derived  from  methylene  blue.  From  this  it  follows  that 
the  introduction  of  the  above-mentioned  acid  group  changes  the  dis- 
tribution in  the  organism  and  causes  especially  a  complete  destruc- 
tion of  neurotropic  properties.  The  central  action  of  a  poison  is  to 
be  explained  logically  by  an  accumulation  of  the  toxic  substance  in 
the  central  nervous  system.  Since,  therefore,  the  central  part  of  the 
toxic  action  has  been  completely  destroyed  by  the  introduction  of  a 
sulfonic  acid  radical  we  find  that  the  reduction  in  toxicity  is  readily 
explained.  It  is  obvious  that  under  these  conditions  other  toxic 
properties,  which  do  not  depend  on  the  central  nervous  system  may 
be  preserved  intact.  Thus  according  to  my  observations  the  blood 
destructive  properties  of  phenylhydrazin  and  benzidin  are  still  present 
in  their  monosulfonic  acids.1 

1  The  action  of  these  combinations  is  not  as  strong  as  the  original  sub- 
stance, but  this  is  probably  due  to  the  fact  that  the  sulfonic  acid  radical  (and 
even  t'he  neutral  sulfonic  radical)  by  itself  reduces  the  toxic  power  of  the  amido 
group.  This  mitigating  action  explains  why  sulfanilic  acid  which  is  derived 
from  anilin  is  no  blood  poison;  this  power  of  the  sulfonic  acid  group,  however, 


CHEMICAL  CONSTITUTION   AND  PHARMACOLOGICAL  ACTION    415 

From  these  considerations  it  is  at  once  clear  that  there  is  a  link 
between  chemical  constitution  and  pharmacodynamic  action,  namely, 
the  distribution  in  the  organism.  In  this  we  are  dealing  with  a  prin- 
ciple which  has  long  been  known,  and  which,  I  might  say,  is  almost 
self-evident,  but  which  nevertheless  is  clearly  expounded  in  but 
few  text-books  on  therapeutics  (see  Stock  vis,  de  Buck,  and  especially 
H.  Schulz). 

Unfortunately  we  have  been  satisfied  with  a  mere  theoretical 
acknowledgment  of  this  principle,  and  have  practically  made  no 
efforts  to  gain  a  deeper  insight  into  the  laws  governing  this  distribu- 
tion. This  is  esepcially  true  of  tne  new  synthetic  tendency,  which 
labors  exclusively  for  symptomatic  effects  and  leaves  questions  con- 
cerning localization  absolutely  untouched.  To  my  mind  just  this 
neglect  is  to  blame  for  the  insufficient  progress  thus  far  made,  and 
I  believe  that  new  points  of  vantage  can  easily  be  gained  if  the 
distributive  views  are  given  greater  prominence.  In  this  connection 
I  may  call  attention  to  the  fact  that  through  the  application  of  the 
principle  of  localization,  which  I  have  attempted,  new  and  promising 
paths  have  been  opened  up  in  the  domain  of  bacteriology,  although 
this  subject  was  already  beginning  to  become  barren  under  the  sche- 
matic application  of  the  doctrines  of  immunity. 

To  be  sure  it  must  be  admitted  that  there  are  enormous  difficulties 
attending  the  determination  of  the  distribution  of  chemical  substances 
with  the  necessary  degree  of  precision.  We  are  here  confronted 
with  a  problem  whose  solution  is  simple  in  only  a  few  special  cases. 
These  we  shall  discuss  in  a  moment.  In  the  great  majority  of 
chemical  compounds,  however,  only  a  combination  of  various  methods 
gives  us  any  definite  knowledge. 

Animal  experiments,  as  such,  do  not  give  us  complete  informa- 
tion concerning  the  distribution  in  the  organism;  they  only  mark 
the  regions  most  susceptible  to  the  poison,  and  then  usually  only  for 
those  systems,  such  as  the  nervous  or  muscular  system,  in  which 
disturbances  of  function  are  recognizable.  The  animal  experiment, 
however,  furnishes  but  little  information  concerning  the  processes  in 
the  vital  parenchyma,  for  to  these  graphic  or  other  ordinary  physio- 
logical methods  are  inapplicable. 

The  assistance  afforded  by  pure  chemical  analysis  is  very  slight. 

is  insufficient  to   destroy  the  powerful  NH-NH2  group  of  phenylhydrazin,  or 
the  two  amido  groups  of  benzidin. 


416  COLLECTED   STUDIES   IN   IMMUNITY. 

It  can  be  carried  out  exactly  with  only  a  very  small  number  of  readily 
determinable  substances,  hence  primarily  with  inorganic  combinations. 
Besides,  the  demonstration  that  a  poison,  for  example  arsenic,  occurs 
in  a  certain  organ,  as  the  brain,  is  of  little  value,  for  this  does  not 
tell  us  what  is  really  of  the  greatest  importance,  namely,  the  localiza- 
tion in  the  separate  cell  constituents  of  the  various  organs. 

The  pathological  and  histological  findings  are  of  far  greater 
importance.  To  be  sure,  if  one  turns  the  pages  of  the  text-books, 
one  will  not  have  very  great  hopes  in  this  direction,  for  the  same 
banal  changes,  fatty  degeneration  of  the  liver,  nephritis,  destruction 
of  the  blood,  are  always  given.  Nissl's  investigations,  however, 
demonstrated  that  exact  histological  studies  on  the  central  nervous 
system  allow  the  points  of  attack  to  be  recognized.  He  showed  that 
certain  poisonings  always  affected  certain  groups  of  ganglion  cells. 
How  fruitful  these  points  of  view  may  be  was  shown  by  the  pretty 
investigations  of  Goldscheider,  through  which  he  showed  that  the 
motor  ganglion  cells  had  already  suffered  demonstrable  lesions  from 
tetanus  poison  at  a  time  when  even  the  slightest  clinical  symptoms 
were  absent.  In  many  other  cases  also,  most  valuable  information 
may  be  furnished  by  minute  histological  investigations;  in  this 
connection  I  may  mention  that  with  cocaine  I  have  found  in  mice 
an  absolutely  specific  foam-like  degeneration  of  the  liver  cells  in  a 
form  which  I  have  seen  with  no  other  substance.  In  general,  I  may 
add  that  the  chronic  poisonings  extending  over  several  days,  and  not 
the  acute  poisonings,  are  best  suited  for  the  demonstration  of  specific 
injuries  to  certain  organs,  a  point  which  has  already  been  emphasized 
by  Nissl. 

In  my  pharmacological  investigations,  which  far  antedate  Nissl's 
publications,  I  have  given  this  method  special  preference.  I  also 
described  a  method  (Deutsche  med.  Wochensch.  1890,  No.  32)  by 
which  these  otherwise  laborious  experiments  can  be  carried  out  with 
ease.  This  method  depends  on  feeding  mice  with  biscuit  which  con- 
tains a  certain  amount  of  the  substance  in  question.  It  is  then  very 
easy  to  find  a  dose  which  will  kill  the  animals  in  the  desired  period 
of  time. 

Although  the  results  of  these  anatomical-pathological  investiga- 
tions are  most  valuable,  it  cannot  be  gainsaid  that  through  them 
one  only  discovers  the  injury  to  the  most  susceptible  organs,  but 
that  the  general  distribution  of  a  certain  substance  within  the  entire 
organism  remains  unknown. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION    417 

In  my  opinion,  however,  this  general  distribution  is  a  very  im- 
portant problem,  for  just  these  facts  furnish  the  most  valuable  in- 
formation concerning  the  chemical  functions  of  the  organs,  and  of 
the  elements  which  compose  them.  At  present  this  problem  can 
only  be  solved  by  the  employment  of  dyes  whose  distribution  we  can 
readily  follow  both  macroscopically  and  microscopically.  It  is  to  be 
deplored  that  these  investigations,  which  possess  such  a  high  didactic 
value  should  thus  far  have  found  so  few  adherents;  they  are  only 
exceptionally  studied  and  then  for  some  particular  purpose. 

If  rabbits  are  injected  with  dyes  it  will  be  found  that  even  macro- 
scopic study  yields  most  interesting  pictures.  There  are  certain 
dyes,  although  not  very  common,  which  stain  only  a  particular  tissue, 
e.g.  fat  tissue;  these  are  called  "  monotropic."  Usually  a  dye 
possesses  an  affinity  for  a  number  of  systems  of  organs,  although 
frequently  it  then  happens  that  one  particular  organ  is  stained  in 
an  especially  conspicuous  manner.  Very  often  one  finds  that  the 
maximum  staining  is  in  the  kidney  (especially  in  the  cortex)  and  in 
the  liver.  Other  dyes,  such  as  acridinorange  and  dimethylamido- 
methylene  blue,  exhibit  their  stain  particularly  in  the  thyroid  gland ; 
still  others,  as  dimethylphenylene  green,  stain  especially  the  fat  tissue; 
some,  such  as  alizarin  blue,  the  submaxillary  gland,  etc. 

Alizarin  blue,  besides  staining  brain  and  kidneys,  stains  the  sub- 
maxillary  gland  with  especial  intensity.  As  examples  of  polytropic 
stains  we  may  mention  neutral  red  and  a  basic  dye,  brilliant  cresyl 
blue,  for  these  stain  the  majority  of  body  parenchyma  intensely  and 
apparently  uniformly.  It  is  particularly  significant  that  the  majority 
of  basic  dyes  which  stain  the  brain  are  also  stored  up  by  fat  tissue. 
As  we  shall  soon  see  neurotropism  and  lipotropism  are  related  to 
one  another. 

The  variation  in  the  localization  of  dyes  frequently  corresponds 
to  certain  peculiarities  in  their  excretion;  the  chief  points  of  excre- 
tion are  probably  kidney  cortex,  liver,  and  intestine.  In  contrast 
to  the  great  majority  of  dyes  which,  like  methylene  blue,  fuchsin, 
alizarin,  indigo  carmine,  and  many  others,  gain  access  to  the  urinary 
secretions  very  easily,  there  are  several  which  seem  incapable  of 
doing  this  and  which  therefore  seem  by  preference  to  be  excreted 
through  the  bile  or  through  the  intestinal  juices.  An  example  of 
this  is  benzopurpurin,  a  very  large-moleculed  cotton  dye  which  is 
made  from  diazotated  toluidin  and  naphylaminsulfonic  acid.1 

1  It  is  possible  that  this  phenomenon  can  be  fully  explained  by  this  that  we 


418  COLLECTED  STUDIES  IN   IMMUNITY 

Besides  this,  however,  one  could  assume  that  analogous  dyes  also 
effect  a  loose  combination  with  the  blood  albumin,  which  makes 
excretion  through  the  kidney  impossible.  In  that  case  the  condi- 
tions would  be  analogous  to  those  which  we  see  with  many  metals, 
e.g.  iron  or  lead,  and  to  those  which  obtain  in  the  excretion  of  a 
poisonous  albuminous  substance,  ricin,  as  they  have  been  deter- 
mined by  investigations  in  the  Pasteur  Institute.  None  of  the  sub- 
stances which  occur  in  the  circulation  in  the  form  of  albumin  com- 
binations pass  into  the  urine,  since  the  albumin  molecule  is  unable 
to  pass  through  the  intact  kidney  filter.  In  contrast  to  this,  how- 
ever, the  intestinal  glands  or  liver  allow  even  these  large-moleculed 
substances  to  pass  through. 

The  salivary  glands  do  not  play  any  important  part  in  elimina- 
tion, as  is  shown  by  the  fact  that  with  the  majority  of  dyes  the  saliva 
is  not  at  all  colored,  and  with  certain  others,  e.g.  alizarin  blue,  is 
but  slightly  tinged.  This  is  apparently  because  of  the  fact  that  the 
salivary  glands  are  not  well  adapted  to  the  secretion  of  substances 
with  large  molecular  weights.  In  the  excretion  of  substances  of 
small  molecular  weights,  however,  they  may  play  a  prominent  role, 
as  can  be  seen  from  the  behavior  of  various  salts,  e.g.,  potassium 
iodide,  rodan  combinations,  and  the  salts  of  mercury.  In  the  aro- 
matic series  it  is  particularly  paraphenylendiamin,  dimethylpara- 
phenylendiamin,  trihydroparaoxychinolin ,  and  related  substances, 
which  are  excreted  through  the  submaxillary  gland  of  rabbits  and 
there  give  rise  to  marked  inflammatory  changes  (oedema,  necrosis). 

The  least  important  role  is  that  taken  by  the  sweat  glands.  So 
far  as  I  am  aware  the  only  dyes  excreted  on  the  body  surface  are 
those  of  the  phosphin  series,  as  is  shown  by  Mannabeig's  researches 
concerning  the  therapeutics  of  malaria. 

Much  greater  significance,  however,  attaches  to  the  possibility  of 
exactly  determining  the  distribution  of  the  dyes  by  means  of  the 
microscope.  I  need  only  call  to  mind  the  vital  staining  of  nerve 
endings  by  means  of  methylene  blue,  a  procedure  which  has  found 


are  here  dealing  with  large-moleculed  substances  which  are  soluble  with  diffi- 
culty and  which  therefore  must  be  regarded  more  like  colloids  In  contrast 
to  methylene  blue,  methyl  violet,  and  many  other  dyes,  benzopurpurin  is  ab- 
solutely non-diffusible.  According  to  the  researches  of  Krafft  (Bericht  der 
deutsch.  chem.^Gesell.  1899)  solutions  of  benzopurpurin  (raising  of  the  boiling- 
point)  showed  an  apparent  molecular  weight  of  3000  instead  of  774  reckoned 
out  from  the  formula. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.  419 

extensive  application  in  the  histology  of  the  nervous  system.  Then 
there  are  the  wonderful  vital  stains  which  the  majority  of  granules 
give  with  neutral  red;  and  the  beautiful  stains  of  these  same 
bodies  which  can  be  effected  with  brilliant  cresyl  blue  (oxazin  dye). 
I  cannot  here  enter  into  still  other  interesting  and  important  vital 
stains. 

Besides  this  each  stain  possesses  its  own  peculiar  characteristics. 
Thus  methylene  blue,  besides  staining  the  nerve  endings  and  a  number 
of  the  most  diverse  granules,  stains  intensely  the  cell  protoplasm  of 
the  islands  of  Langerhans  of  the  pancreas,  and,  further,  also  muscle 
cells  of  a  certain  particular  function,  striped  as  well  as  smooth.  I 
am  practically  convinced  that  in  the  vascular  system  certain  muscle 
fibres  which  can  be  stained  with  methylene  blue  cause  a  marked 
narrowing  and  perhaps  even  a  complete  closure  of  the  lumen  after 
the  manner  of  a  ligature.  These  muscle  fibres  never  form  a  con- 
tinuous lining  of  the  vessel  wall  but  only  occur  singly  and  separated 
from  one  another  by  comparatively  wide  intervals.  The  uniform 
calibration  of  the  tube  would  then  fall  to  the  lot  of  the  evenly  dis- 
tributed muscle  lining  which  takes  no  stain.  We  should  thus  have 
what  is  surely  of  great  significance,  namely,  the  fact  that  vessel 
calibration  and  vessel  closure  are  two  functions  which  are  absolutely 
distinct  anatomically  and  biologically.  In  a  description  so  general 
in  character  as  this  one  I  cannot  enter  into  still  other  interesting 
groups  of  dyes,  e.g.,  those  that  stain  nuclei  vitally,  etc. 

Exactly  the  same  differences  which  we  have  observed  in  the  case 
of  dyes  manifest  themselves  if  we  introduce  other  kinds  of  sub- 
stances into  the  body,  it  matters  not  whether  they  are  well  defined, 
organic  or  inorganic  combinations,  or  whether  they  constitute  chem- 
ically unknown  and  highly  complex  bacterial  products.  In  general 
we  shall  probably  have  to  assume  that  substances  which  are  chemically 
tvell  defined  are  to  a  great  extent  polytropic  in  character.  In  my 
studies  with  several  substances  readily  demonstrable  by  means  of  color 
reactions  and  whose  distribution  can  therefore  readily  be  followed, 
I  have  convinced  myself  that  the  aromatic  bases  as  a  rule  have  an 
affinity  for  many  different  kinds  of  parenchyma.  If  in  spite  of  this 
the  clinical  injury  manifests  itself  in  only  one  tissue,  this  in  no  way 
contradicts  the  polytropic  character  of  these  substances.  It  merely 
proves,  what  is  really  a  matter  of  course,  that  among  a  number  of 
tissues  there  are  some  that  are  particularly  susceptible  to  an  equal 
injury.  To  what  extent  other  circumstances,  such  as  saturation  of 


420  COLLECTED  STUDIES  IN   IMMUNITY 

the  tissues  with  oxygen,  reaction  of  the  tissues  (nephritis  in  chromium 
poisoning),  conditions  of  alkalinity,  peculiarities  of  elimination,  etc  , 
affect  the  result  in  any  given  case  cannot  now  be  discussed.  We 
find  exactly  the  same  conditions  to  hold  with  bacterial  poisons. 
Tetanus  poison,  for  example,  as  is  shown  by  the  experiments  of 
Ddnitz,  Roux,  and  others,  is  monotropic  in  highly  susceptible  animals, 
whereas  in  other  animals,  rabbits,  pigeons,  etc.,  the  tetanus-binding 
groups  are  present  not  only  in  the  brain  but  also  in  a  number  of  other 
organs  of  less  biological  importance.  This  explains  why,  for  instance, 
in  guinea-pigs  the  lethal  dose  is  the  same  whether  the  poison  is  in- 
jected subcutaneously  or  intracerebrally,  whereas  in  the  pigeon, 
and  to  a  certain  extent  also  in  the  rabbit,  much  larger  doses  are 
required  for  subcutaneous  poisoning.  Under  these  circumstances 
part  of  the  poison  is  laid  hold  of  by  the  body  parenchyma  and  thus 
deflected  from  the  endangered  organs. 

We  may  perhaps  regard  it  as  a  matter  of  course,  that  these  laws 
of  mutual  deflection  play  an  important  role  in  all  polytropic  sub- 
stances, and  that  we  shall  gain  a  real  insight  into  the  action  of  drugs 
only  if  we  regard  this  factor  sufficiently.  If,  for  instance,  as  is  so  often 
the  case,  a  poison  is  both  neurotropic  and  lipotropic,  if  the  same 
amount  of  poison  per  kilo  body  weight  is  injected  into  a  lean  animal 
as  into  a  very  fat  one,  it  is  clear  that  the  share  of  poison  which  falls 
upon  the  brain  in  the  former  case  is  much  greater  than  in  the  latter. 

IV. 

We  now  take  up  the  question  as  to  how  this  varied  distribution 
occurs.  As  a  rule  the  poisons  reach  the  tissues  through  the  circu- 
lation, and  we  shall  therefore  first  study  the  influence  of  the  vascular 
system  on  this  distribution.  A  moment's  consideration,  however, 
shows  that  although  the  circulation  may  be  the  prerequisite,  it  can 
in  no  way  be  the  cause  of  the  varied  distribution  discussed  above. 
According  to  the  views  held  by  the  majority  of  investigators  and 
also  by  me  this  localization  in  certain  organs  depends  in  every  in- 
stance on  causes  within  the  tissues  and  not  on  the  vascular  distri- 
bution. For  example,  if  in  a  case  of  jaundice  we  find  that  the  brain 
shows  not  a  trace  of  bilirubin  coloration,  while  many  other  tissues, 
such  as  kidney,  liver,  etc.,  are  saturated  with  bile  pigment,  this,  in 
my  opinion,  is  due  to  the  chemistry  of  the  brain  substance.  The 
brain  lacks  all  such  substances  which  attract  bilirubin,  that  is  to 


CHEMICAL   CONSTITUTION  AND  PHARMACOLOGICAL  ACTION    421 

say  bilirubin  is  not  neurotropic.  In  recent  years  a  different  view 
has  been  promulgated,  especially  by  Biedl,  who  ascribes  a  decisive 
role  in  the  distribution  of  poisons  to  the  vessel  wall.  As  a  result 
of  my  own  long  experience  with  the  greatest  variety  of  substances 
I  am  unable  to  assume  that  the  vascular  endothelium  as  such  exer- 
cises different  functions  in  different  organs,  so  that,  for  example, 
a  liver  capillary  is  permeable  for  certain  substances  which  will  not 
pass  through  other  capillaries.1 

On  the  other  hand  the  vascular  system  plays  a  very  important 
role  in  a  different  direction,  as  can  be  seen  from  the  following  strik- 
ing example.  Mice  are  fed  according  to  my  "biscuit  method"  with 
derivatives  of  paraphenylendiamin  (acetylparaphenylendiamin,  thio- 
sulfonic  acid  and  mercaptan  of  paraphenylendiamin).  On  autopsying 
the  animals  very  peculiar  changes  are  observed  in  the  diaphragm. 
The  parts  surrounding  the  central  tendon  are  stained  intensely  brown, 
while  the  peripheral  portions  are  usually  unstained.  Frequently 
the  margin  of  the  stain  is  wavy  and  marked  by  a  more  intense  colora- 
tion. At  times  I  have  observed  similar  changes  in  other  muscular 
regions,  namely,  in  those  of  the  eye,  larynx,  and  tongue.  Micro- 
scopical examination  shows  that  this  is  not  a  case  of  infarct,  but 
that  there  is  apparently  a  uniform  brown  staining  of  the  muscle 
areas  in  question.  The  cross  stnation  is  preserved  intact,  and  a 
moderate  degree  of  fatty  degeneration  is  not  infrequently  observed. 
"Usually  also  there  is  a  certain  amount  of  hyperaemia.  We  are  not 
dealing  with  a  derivative  of  hemoglobin;  on  the  contrary  it  is  much 
more  probable  that  we  are  dealing  with  a  highly  complex  oxidation 
product  of  the  paraphenylendiamin.2 

The  question  which  now  arises  is  why,  in  this  feeding,  only  part 
of  the  muscles,  a  very  small  part,  show  this  vital  staining. 

It  was  soon  seen  that  the  groups  of  muscles  affected  were  analo- 
gous in  other  respects.  Thus  with  injections  of  methylene  blue  it 

1  It  was  especially  gratifying  to  note  that  Bruno,  as  a  result  of  the  investi- 
gations which  he  made  under  the  direction  of  R.  Gottlieb,  is  also  very  skeptical 
regarding  Biedl's  views  (Deutsche  med.  Wochensch.  1899,  No  23). 

•This  assumption  has  subsequently  been  clearly  confirmed  by  the  work  of 
Dr.  Rebnp  (Archiv  internat.  de  Pharmacodynamie,  Vol.  VIII,  p.  203)  It 
was  found  in  animals  poisoned  acutely  with  parapbenylendiamin  that  the 
muscles  which  were  saturated  with  the  poison  assumed  the  typical  brown  color 
when  brought  in  contact  with  air.  I  would  also  call  attention  to  the  fact  that 
both  paraphenylendiamin  and  paramidophenol  are  employed,  by  oxidation, 
for  true  brown  and  black  dyes  for  hair  and  fur  (Ursol  dye) 


422  COLLECTED   STUDIES  IN  IMMUNITY, 

is  just  in  these  areas  that  the  motor  nerve  endings  take  a  more  or 
less  complete  stain.  In  comparative  pathology  also  we  find  this 
group  in  evidence,  for  trichinae  invade  by  preference  diaphragm,  and 
the  muscles  of  the  eye  and  larynx. 

These  facts  are  very  readily  explained.  In  accordance  with  a 
principle  discovered  by  Robert  Mayer,  the  blood-supply  of  the  muscles 
is  dependent  on  their  biological  importance.  Muscles,  such  as  the 
diaphragm,  which  labor  continuously  and  whose  failure  to  act  would 
constitute  a  marked  disturbance  of  health  are  far  better  supplied 
with  blood  than  others  of  less  importance. 

Naturally  in  this  group  of  "most  favored"  muscles,  correspond 
ing  to  the  greater  supply  of  blood,  there  will  also  be  a  maximum 
supply  of  oxygen,  foodstuffs,  and  all  other  materials  present  in  the 
circulation.  Hence  such  a  muscle  cell  will  be  more  highly  charged 
with  oxygen  and  can  therefore  exert  a  more  energetic  oxidizing 
action,  as  is  manifested  in  the  brown  staining  with  paraphenylen- 
diamin.  The  staining  of  the  muscle  end-plates  is  explained  in  exactly 
the  same  way,  through  the  increased  supply  of  methylene  blue  on 
the  one  hand,  and  the  saturation  with  oxygen  and  the  alkaline  con- 
stitution of  the  nerve  endings  on  the  other. 

An  important  principle  governing  the  distribution  of  substances 
in  the  organism  can  be  deduced  for  these  experiments,  namely,  that 
myotropic  and  neurotropic  substances  can  produce  an  isolated  injury 
to  certain  systems  solely  through  the  character  of  the  blood-supply. 
It  would,  however,  be  wrong  to  assume  that  all  muscle  and  nerve 
poisons  must  always  injure  only  the  most  favored  system  of  muscles 
as  described  above.  That  would  be  disregarding  the  fact  that  the 
poisonous  action  is  dependent  not  only  on  the  supply  of  poisons 
but  also  on  the  capacity  of  the  tissues  to  take  up  the  poison.  A 
nerve  ending  of  neutral  or  acid  reaction  will  take  up  other  substances 
(e.g.  alizarin)  than  one  of  alkaline  reaction  (methylene  blue) ;  a 
muscle  loaded  with  oxygen  will  oxidize  certain  substances  and  so 
overcome  their  poisonous  action,  whereas  this  same  poison  will  re- 
main intact  in  muscle  tissue  deficient  in  oxygen. 

I  believe  that  the  various  nerve  endings— motor,  sensory,  and 
secretory — are  made  up  of  the  same  chemical  material.  If,  however, 
we  consider  the  manifold  and  specialized  actions  of  the  alkaloids, 
for  example,  the  very  different  actions  of  digitalis,  curare,  pilocarpin, 
and  atropin,  and  if  we  ascribe  the  toxic  action  to  an  accumulation, 
we  shall  be  forced  to  conclude  that  the  nerve  endings,  though  com- 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION    423 

posed  of  the  same  chemical  substances,  are  subjected  to  different 
conditions  in  the  various  tissues,  conditions  which  may  possess  a 
decisive  influence.  Foremost  among  these  I  regard  variations  in  the 
reaction  and  in  the  degree  of  oxygen  saturation  to  which  I  have 
already  referred.  As  a  result  of  my  experiments  in  biological  stain- 
ing I  assume  that  certain  nerve  endings,  central  and  peripheral,  are 
characterized  by  a  particular  complex  of  such  determining  factors, 
and  that  this  "  chemical  milieu  "  represents  the  resultant  of  the  normal 
physiological  functions.  Whether  these  views  possess  any  heuristic 
value  for  the  further  development  of  the  science,  I  do  not  know. 
For  the  present  I  shall  content  myself  by  remarking  that  the  isolated 
disease  of  nerve  or  muscle  apparatus,  so  far  as  it  affects  certain  par- 
ticular groups  (lead  paralysis,  arsenic  paralysis),  is  readily  explained 
from  this  point  of  view.  We  shall  have  to  assume  the  existence  of 
just  as  many  different  types  of  nutrition  as  we  can  demonstrate 
different  types  of  disease. 

This  brings  me  to  a  further  question  which  concerns  this  dis- 
tributive therapy,  and  that  is  whether  it  is  possible  simply  by  chem- 
ical means  to  change  the  type  of  distribution  of  a  given  substance. 
This  question  can  readily  be  answered  in  the  affirmative.  If,  for 
example,  a  frog  is  injected  with  methylene  blue,  the  nerve  endings, 
as  is  well  known,  will  be  stained  in  the  living  state.  However,  if 
an  easily  soluble  acid  dyestuff,  e.g.  orange-green,  is  added  to  the 
methylene  blue  solution  so  that  a  clear  green  solution  results,  it 
will  be  found  that  the  injection  of  such  a  mixture  no  longer  produces 
staining  of  the  nerve  endings.  Hence  we  see  that  the  conditions  are 
entirely  analogous  to  those  which  we  find  in  the  staining  of  dry  prepa- 
rations. The  basic  dyes  by  themselves  stain  nuclei,  whereas  the 
combination  of  basic  dyes  writh  acid  dyes,  which  I  introduced  into 
histological  technique  under  the  name  of  "triacid  dyes,"  lack  this 
property  to  a  greater  or  less  degree.  In  both  cases  we  are  dealing 
with  a  distribution  of  the  methylene  blue  between  the  acid  dye  and 
the  tissue  constituents.  The  tissues  as  well  as  the  acid  dyestuff  have 
an  affinity  for  the  methylene  blue.  If  the  affinity  of  the  tissues  is 
greater,  they  will  be  stained  blue;  if  that  of  the  acid  dye  is  the  greater, 
the  staining  will  not  occur.1 


1  Naturally  this  phenomenon  will  occur  conspicuously  only  in  those  cases 
in  which  the  tissue  substances  possess  an  affinity  for  the  base  only  and  not  for 
the  acid  dye  If  the  latter  condition  obtains  the  mixture  of  both  components 


424  COLLECTED  STUDIES  IN   IMMUNITY. 

In  the  deflection  of  methylene  blue  by  means  of  orange  we  thus 
have  presented  a  phenomenon  which  in  its  essential  features  reminds 
us  of  the  mode  of  action  of  the  antitoxins. 

The  opposite  hehavior,  however,  also  occurs,  namely,  that  the 
localization  of  a  certain  substance  in  a  particular  tissue  becomes 
possible  only  through  the  simultaneous  introduction  of  a  second 
combination,  even  though  the  latter  effects  no  union  whatever  with 
the  first  combination.  Naturally  these  complicated  phenomena  can 
be  demonstrated  with  certainty  only  by  the  aid  of  vital  stain  ings,  foj 
in  these  can  the  microscopical  distribution  be  positively  determined. 
The  following  examples  are  the  result  of  this  method  of  investigation : 

Bismarck  brown,  the  well-known  basic  azo  dye,  exhibits  a  certain 
amount  of  neurotropy  manifested  especially  in  the  staining  of  the 
gray  matter  of  the  brain.  This  affinity,  however,  is  insufficient  to 
give  rise  to  a  staining  of  the  peripheral  nerve  endings  in  a  frog, 
particularly  a  staining  of  the  taste  bulbs.  If,  however,  a  frog  is 
injected  with  a  mixture  of  methylene  blue  and  Bismarck  brown 
it  will  be  found  that  the  terminal  apparatus  is  stained  a  mixed 
shade.  The  blue  very  readily  loses  its  color  through  reduction,  and 
in  a  preparation  mounted  on  a  slide  and  sealed  with  a  cover-glass 
the  blue  color  can  be  seen  to  disappear  rapidly,  leaving  only  a  pure 
brown  stain. 

The  other  example  is  still  more  striking:  If  a  rabbit  is  infused 
with  a  solution  of  methylene  blue,  one  always  finds  well-marked  stain- 
ing of  the  pancreas,  due  especially  to  a  staining  of  the  granules  and 
protoplasm  of  the  islands  of  Langerhans.  In  no  case  have  I  ob- 
served a  staining  of  the  nerve  endings  under  these  conditions.  If, 
however,  one  adds  certain  dyestuffs  of  the  triphe'nylmethane  series 
to  the  fluid  infused,  dyes  which  in  themselves  do  not  stain  the  nerve 
endings,  a  truly  beautiful  staining  of  the  nerve  apparatus  frequently 
occurs.  In  these  and  other  similar  cases  I  believe  that  we  can 
only  assume  that  the  favoring  substances  cause  a  modification  of  the 
function  of  the  apparatus  in  question,  and  that  this  carries  with  it  a 
change  in  the  "chemical  milieu  "  defined  above,  and  so  in  the  ab- 
sorbing power.  It  is  possible  that  similar  factors  also  play  a  certain 
r61e  in  many  abnormal  actions  of  drugs,  especially  in  inherited  or 
acquired  hypersensitiveness. 


(i.e.  the  neutral  stain)  will  come  into  play,  a  fact  which  is  so  well  observed  in 
the  staining  of  the  neutrophilic  granules. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.  425 


V. 

The  question  now  arises  as  to  how  we  conceive  this  selection  of 
the  tissues  to  occur.  It  is  very  probable  a  priori  that  we  are  dealing 
with  chemical  affinities  in  the  widest  meaning  of  the  term.  We  must, 
however,  discuss  in  detail  the  nature  of  these  affinities.  In  this, 
I  must  emphasize,  we  are  dealing  primarily  with  substances  which, 
like  the  various  natural  and  artificial  drugs,  are  foreign  to  the  body, 
not  with  foodstuffs  capable  of  assimilation.  The  latter  will  be 
treated  by  themselves  subsequently. 

The  simplest  case  is  that  in  which  the  organism  is  injected  with 
indifferent  substances,  neither  acid  nor  basic  in  character,  to  which, 
corresponding  to  their  constitution,  we  can  ascribe  no  great  chemical 
affinities,  but  which  nevertheless  exert  marked  and  often  highly 
toxic  effects.  In  this  category  belong  especially  the  various  hydro- 
carbons, e.g.  toluol,  benzol;  a  number  of  ketones,  such  as  acetophe- 
non ;  many  sulfones,  which  are  characterized  by  their  chemical  in- 
difference; also  various  kinds  of  ethers,  alcohols,  and  a  large  number 
of  other  narcotics.  The  best  opinion  seems  to  be  that  in  these 
cases  no  direct  chemical  affinities  come  into  play  on  the  part  of  the 
organism,  and  that  the  molecule  is  always  present  in  the  tissue 
constituents  unchanged  and  chemically  uncombined.  That  is  to 
say,  the  phenomenon  is  one  of  contact  action.  In  spite  of  this  it 
can  readily  be  shown  that  all  these  compounds  possess  a  typical 
localization  in  the  tissues,  the  cause  of  which  we  shall  soon  discuss. 

First,  however,  I  should  like  to  say  a  few  words  concerning  the 
historical  side  of  this  question.  The  idea  that  chemical  substances 
can  act  solely  through  contact  was  first  affirmed  many  years  ago, 
thus  by  Buchheim  in  1859,  Schmiedeberg  in  1883,  Harnack  in  1883, 
and  by  Geppert.  The  latter 's  investigations  may  be  found  in  the 
Zeitschrift  fur  klin.  Medicin,  Vol.  XV,  and  deal  with  the  nature  of 
prussic-acid  poisoning.  He  showed  that  in  this  highly  interesting 
case  the  hydrocyanic  acid  acts  as  such.  He  explained  the  result  of 
the  toxic  action  in  the  following  manner: 

"  We  know  that  chemical  processes  are  retarded  simply  through  the 
presence  of  minimal  amounts  of  prussic  acid.  Thus  iodic  acid  does 
not  yield  up  its  oxygen  to  formic  acid  under  conditions  otherwise 
favorable  if  even  a  minimal  amount  of  prussic  acid  is  present.  It 
is  quite  natural,  I  suppose,  that  in  the  poisoned  organism,  highly 


426  COLLECTED  STUDIES   IN   IMMUNITY 

oxidized  substances  (the  analogues  of  iodic  acid)  are  no  longer  able 
to  yield  up  their  oxygen  to  oxidizable  combinations  when  prussic 
acid  is  present.  (One  must  think  of  these  highly  oxidized  substances 
as  transmitters  or  carriers  of  oxygen.)  Prussic  acid  poisoning  is 
therefore  an  internal  suffocation  of  the  organs." 

This  discovery  of  contact  action  constituted  the  first  step  toward 
penetrating  the  mystery  of  the  action  of  drugs.  This,  however, 
afforded  no  explanation  as  to  why  the  substances  mentioned  ex- 
hibited an  elective  action.  That  was  because  the  link  was  missing 
which,  according  to  modern  views,  is  absolutely  indispensable,  namely 
the  connection  between  action  and  distribution  in  the  tissues.  I 
think  I  am  justified  in  claiming  to  be  the  first  to  recognize  the  right 
path,  for  in  1887,  in  my  article  on  "  The  Therapeutic  Significance 
•of  the  Substituting  Sulphuric  Acid  Group"  (Therap.  Monatshefte, 
March,  1887),  I  demonstrated  that  neurotropic  stains  are  deprived 
of  this  property  on  the  addition  of  the  sulfonic-acid  group.  Even  at 
that  time  I  compared  the  localization  of  the  dyes  and  of  the  alkaloids 
in  the  brain  with  the  principle  of  the  shaking-out  procedure  devised 
by  Stas-Otto,  expressing  myself  as  follows: 

"The  principle  of  'shaking-out'  poisons  devised  by  Stas-Otto 
depends  on  the  fact  that  basic  substances,  e.g.  alkaloids,  etc.,  are 
generally  firmly  combined  in  acid  solutions,  and  hence  extracted 
with  difficulty,  whereas  the  same  substances  can  readily  be  shaken 
out  of  alkaline  solutions.  Acid  substances,  of  course,  exhibit  exactly 
the  opposite  behavior:  they  are  held  back  by  alkaline  media,  but 
readily  given  up  by  acid  media.  If  we  apply  these  experiences  to 
the  question  under  discussion  we  can  readily  understand  why  basic 
dyes  (which  are  not  held  back  by  the  blood  through  any  chemical 
affinities)  are  especially  laid  hold  of  by  the  brain,  whereas  the  acid 
dyes  and  the  sulfonic  acids  (which  are  bound  by  alkalies  of  the  blood 
to  form  salts,  and  are  thus  anchored,  as  it  were)  show  exactly  the 
opposite  behavior." 

Besides  this  I  showed  that  fat  tissue  behaves  like  the  brain, 
for  a  large  part  of  the  substances  taken  up  by  the  brain  are  taken 
up  also  by  the  fat  tissue.  In  1891  this  question  received  a  fresh 
impetus,  for  Hofmeister,  Pohl,  and  also  Spiro,  called  attention  to 
the  significance  of  loose  combinations  which  could  readily  be  dis- 
sociated. Thus  in  1891  Pohl  showed  that  the  ability  of  the  red 
blood-cells  to  take  up  chloroform,  a  fact  which  Schmiedeberg  had 
demonstrated  in  1867,  was  due  to  the  cholesterm  and  lecithin  which 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.    427 

the  cells  contain.  Both  substances  can  be  shaken  out  with  chloro- 
form. He  also  referred  the  union  of  chloroform  in  the  brain  to 
similar  fat-like  bodies  in  that  organ,  as  I  have  done  for  the  color- 
ing matter  of  the  alkaloids.  A  basis  was  thus  secured  from  which  to 
study  the  action  of  the  above-mentioned  substances  in  the  brain. 
These  substances,  it  will  be  seen,  are  most  all  readily  soluble  in  fats 
and  fat-like  bodies,  corresponding  to  their  physico-chemical  nature.1 

The  conditions,  howrever,  were  far  more  complex  in  the  large 
number  of  bodies  which,  like  many  medicinal  substances  (e.g.,  the 
antipyretics),  and  the  most  varied  basic  substances  (among  these  the 
alkaloids),  phenols,  aldehydes,  and  many  others,  in  contrast  to  the 
indifferent  bodies,  do  not  seem  incapable  of  combining  synthetically 
with  the  tissues.  In  numerous  articles  Low  assumes  that  most  of  the 
bodies  in  question  are  able  to  unite  synthetically  with  constituents 
of  the  cell  or  with  the  living  protoplasm.  It  is  obvious  that  we  must 
assume  the  protoplasm  to  contain  many  different  kinds  of  atomic 
groups  possessing  very  strong  affinities,  and  it  was  certainly  very 
plausible  when  Low  ascribed  a  leading  role  in  the  phenomena  of 
poisoning,  to  groups  so  well  able  to  act.  His  experiments  and  re- 
searches lead  him  to  conclude  that  in  the  cell  it  is  particularly  alde- 
hyde groups  or  labile  amido  groups  which  play  this  anchoring  or 
grasping  role.  According  to  Low  all  substances  which  can  combine 
with  these  two  radicals  are  poisons  for  the  protoplasm;  the  greater 
the  affinity  the  stronger  the  poisonous  action. 

Against  this  view  of  a  substituting  action  of  the  poisons  a  large 
number  of  easily  verified  facts  can  be  brought  forward.  If  benzalde- 
hyde  and  anilin  (or  phenylhydrazin,  etc.)  are  mixed,  the  two  sub- 
stances will  condense  to  form  a  new  substance,  benzylidenanilin, 
water  separating  at  the  same  time.  This  benzylidenanilin  is  a  single 

1  It  is  impossible  to  do  more  than  refer  to  the  great  advances  made  since 
my  address,  especially  through  the  labors  of  Hans  Meyer  and  Overton.  iu 
three  studies  on  the  theory  of  alcohol  narcosis  (Archiv  f  expenm.  Pathologic 
1899-1901),  Meyer  has  shown  in  the  most  exact  manner  for  a  large  number 
of  chemical  substances  that  the  mode  of  action  of  the  indifferent  narcotics 
is  not  dependent  on  their  other  chemical  properties  but  is  governed  exclu- 
sively by  the  partition  coefficient  which  determines  their  distribution  among 
water  and  certain  fat -like  substances  (brain  and  nerve  fat),  H  Overton  came 
to  the  same  conclusion  regarding  the  causal  relation  between  solubility  in  fat 
and  narcotic  action.  His  investigations,  \\hich  have  been  gathered  together  m 
a,  work  entitled  "Studien  iiber  die  Xarkose,"  Jena,  1901,  dealt  especially  with 
vegetable  cells  and  small  animals  present  in  the  fluid. 


428  COLLECTED  STUDIES  IN  IMMUNITY. 

body  which  does  not  give  up  either  anilin  or  benzaldehyde  to  indif- 
ferent solvents.  It  requires-  chemical  splitting  in  order  to  form  the 
two  original  substances. 

In  this  way  the  question  can  very  readily  be  decided  whether  or 
not  a  certain  substance  is  anchored  to  a  cell  synthetically,  for  the 
material  in  question  need  simply  be  treated  with  indifferent  solvents 
possessing  strong  extractive  properties  (alcohol,  ether,  etc.).  If 
animals  are  injected  with  the  most  varied  poisons,  alkaloids,  phenols, 
anilin,  dimethylparaphenylendiamin,  antipyrin,  thallin,  etc.,  and  if 
one  waits  until  the  distribution  is  completed  (which  usually  occurs 
in  a  moment),  it  is  easy  to  extract  the  unchanged  poison  by  means 
of  suitable  methods  of  extraction,  and,  provided  the  substance  is  easily 
detected,  like  thallin  or  dimethylparaphenylendiamin,  to  discover  it  in 
the  tissues  by  means  of  staining  reactions.  Naturally  these  experi- 
ments are  carried  out  most  strikingly  with  dyestuffs,  for  in  these  the 
extractive  decolorization  of  the  methylene-blue  brain  cortex  or  of  the 
fuchsin  kidney  can  very  easily  be  followed. 

The  experiments  with  dyestuffs  furnish  still  another  argument 
against  a  process  of  substitution.  In  the  basic  dyes  when  one  or 
several  amido  groups  are  replaced  by  aldedyde  radicals  a  change  in 
color  often  takes  place.  Thus  by  means  of  aldehyde,  fuchsin  red 
is  made  to  yield  violet  dyes.  In  accordance  with  Low's  theory  one 
would  have  been  led  to  suppose  that  when  suitable  dyestuffs  were 
employed  a  change  of  color  due  to  substitution  should  occur  in  some 
case  or  other  and  in  some  organ  or  other.  In  spite  of  experiments 
specially  devised  for  the  purpose  I  have  never  observed  this  to  occur, 
either  with  dyestuffs  which,  like  those  mentioned  above,  unite  with 
aldehyde,  or  with  certain  basic  dyes  (e.g.,  the  azonium  base  which 
Kehrmann  produces  from  safranin)  which  take  up  amido  radicals 
of  the  most  varied  kinds  and  cause  an  intensification  and  change  of 
the  color  characteristics. 

Many  other  reasons  can  be  adduced  which  speak  against  the 
correctness  of  Low's  theory.  I  may  merely  mention  the  transitory 
character  of  the  action,  a  point  which  is  so  often  noted,  especially  in 
the  alkaloids;  furthermore,  in  the  case  of  many  drugs,  the  rapid 
elimination,  which  argues  against  a  firm  synthetic  combination; 
another  fact,  one  which  may  perhaps  be  of  practical  importance,  is 
this:  that  in  the  construction  of  new  therapeutic  substances  efforts 
were  directed  particularly  to  the  elimination  (by  appropriate  sub- 
stitution) of  groups  which  could  effect  syntheses.  This  is  the  case, 


CHEMICAL   CONSTITUTION'  AND  PHARMACOLOGICAL  ACTION.      429 

for  example,  with  phenacetin,  in  which  by  the  introduction  of  the 
methyl  radical  and  of  the  acetyl  group  the  powerful  OH  and  NH2 
groups  of  paramidophenol  are  occupied. 

All  this  has  led  me  to  conclude  positively  that  Low's  theory  of 
the  substituting  action  of  therapeutic  substances  is  untenable. 

By  this  I  do  not  in  the  least  wish  to  say  that  groups  capable  of 
reacting,  such  as  Low  presupposes  to  exist  in  the  living  protoplasms, 
cannot  occur  there.  It  must  be  borne  in  mind,  however,  that 
condensation  phenomena  are  not  produced  merely  by  the  presence 
of  two  substances  capable  of  condensing,  but  that  the  combining 
affinity  must  usually  first  be  increased  through  appropriate  means, 
such  as  increase  of  temperature,  the  addition  of  substances  abstract- 
ing water,  etc.  Even  in  the  practice  of  the  synthetic  chemist,  who 
allows  the  substances  to  act  on  one  another  either  directly  or  in  con- 
centrated solutions,  such  direct  condensations  are  not  especially  fre- 
quent. The  number  of  these,  however,  is  still  more  limited  if  the 
synthesis  is  to  occur  under  conditions  corresponding  to  those  in  the 
living  organism,  i.e.  in  dilute  solutions,  at  low  temperature  and  in 
the  absence  of  suitable  auxiliary  substances.  Dimethylamidoben- 
zaldehyde  unites  with  indol,  for  example,  even  in  dilute  solutions, 
at  room  temperature,  forming  a  red  dye,  but  only  when  the  solution 
contains  small  amounts  of  f  ee  mineral  acid.  If  this  is  absent,  or  if 
the  solution  is  even  faintly  alkaline,  no  combination  of  any  kind 
occurs. 


VI. 

These  considerations  lead  at  once  to  the  view  that  in  certain 
cases  apparently  it  still  is  possible  to  effect  a  substitution  within  the 
organism  by  the  introduction  of  chemical  substances.  In  order  to 
accomplish  such  a  synthesis  the  selection  of  suitable  substances  will 
be  prerequisite,  and  these  substances  must  be  of  such  a  chemical 
constitution  that  they  can  exert  chemical  influences  of  the  most 
powerful  kind.  I  have  made  extensive  experiments  with  many 
hundreds  of  different  combinations,  and  in  all  of  these  I  have  only 
discovered  one  substance  to  which  I  am  inclined  to  ascribe  such 
a  substituting  action  on  protoplasm.  This  substance,  vinylamin, 
discovered  by  Gabriel  and  described  by  him  in  a  masterly  manner, 
is  formed  by  abstracting  bromine  from  bromethylamine  by  means  of 
potassium. 


430  COLLECTED  STUDIES  IN   IMMUNITY. 

CH2 

Bromethylamine  = 


Since  then,  however,  Marckwald  has  positively  shown  (1900- 
1901)  that  this  substance  cannot,  as  was  at  first  supposed,  contain  a 
double  bond  (ethylene  combination),  for  it  does  not  reduce  per- 
manganate at  ordinary  temperature  nor  take  up  bromine.  It  can 
therefore  only  possess  the  constitution  of  a  dimethylenimin: 

>N>NH 
H2/ 

In  view  of  this  a  complete  analogy  exists  between  the  ethylenimin 
and  the  ethylenoxid: 

CH2v 
i       \n 


C 


In  conformity  with  Bayer's  tension  theory  we  must  ascribe  an 
extraordinary  tension  to  the  three-sided  ring  contained  in  the  di- 
methylenimin. This  manifests  itself  also  in  the  fact  that  this  sub- 
stance shows  a  marked  tendency,  through  the  addition  of  acid  radicals 
and  the  breaking  of  the  ring,  to  pass  over  into  a  substituted  ethyl - 
amin  of  the  chain  series.  Thus,  as  Gabriel  showed,  HC1  is  added  with 
the  formation  of  chlorethylamin,  and  sulphurous  acid  with  the  forma- 
tion of  taurin.  These  reactions  proceed  with  great  energy,  as  is 
shown  by  the  fact  that  even  in  dilute  watery  solutions  of  the  freshly 
prepared  hydrochloride  an  alkaline  reaction  develops  within  a  few 
minutes,  due  to  the  formation  of  free  chlorethylamin  which  reacts 
alkaline. 

Ethylenoxid  behaves  in  an  analogous  manner.  This  is  shown 
in  surprising  fashion  by  the  fact  that  this  neutral  body  precipitates 
magnesia  out  of  chlormagnesium,  iron  oxide  out  of  iron  chloride, 
entirely  after  the  manner  of  free  alkalies.  In  doing  so  it  adds  the 
acid  radical  and  becomes  transformed  into  chlorethylalcohol. 

These  two  substances,  ethylenimin  and  ethylenoxid,  are  highly 
toxic  combinations  as  has  been  shown  by  the  researches  of  Levaditi 
and  myself.  The  pathological  changes  excited  by  dimethylenimin 

1 1  have  taken  the  liberty  of  somewhat  modifying  the  text  of  this  chapter 
in  accordance  with  the  positive  advance  of  our  knowledge,  which  we  owe  to 
the  labors  of  Marckwald. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL   ACTION      431 

are  especially  interesting.  Administered  to  a  great  variety  of  ani- 
mals (mouse,  rabbit,  dog,  goat,  guinea-pig,  rat)  in  doses  which  cause 
death  after  1^  to  2  days  or  more,  this  substance  causes  total  necrosis 
of  the  kidney  papilla.  In  the  rabbit  Levaditi  found,  besides  this, 
marked  changes  extending  from  the  pelvis  of  the  ureter  to  the  urethra, 
and  consisting  of  necrosis  of  the  lining  epithelium,  hemorrhages,  and 
oedema.  (Archives  internat.  de  pharmacodynamie,  Vol.  VIII,  1901.) 

Every  one  who  has  learned  to  know  these  changes — changes 
absolutely  unique  in  pathology— will  be  forced  to  the  assumption 
that  this  localization  is  dependent  on  a  direct  attack  of  the  vinylamin 
on  the  affected  epithelia,  an  ethyl  amido  group  entering  the  proto- 
plasmic molecule.  This  assumption  is  supported  by  the  fact  hat 
only  the  active  three-sided  ring  is  able  to  produce  this  phenomenon, 
not  the  ethylene  combination  (CH2=rCH2),  furthermore,  the  fact  that 
neunn  (trimethylvinylammonium  hydroxid)  which  can  be  obtained  by 
an  exhaustive  methylation  of  the  dimethylenimin,  acts  in  an  entirely 
different  manner.  That  we  are  dealing  with  a  typical  ethylene  com- 
bination is  shown  by  the  behavior  toward  bromine  and  permanganate 
of  potash. 

It  has,  of  course,  long  been  known  that  neurin  is  a  highly  toxic 
substance.  Aside  from  its  clinical  toxicological  mode  of  action  it 
is  characterized  by  an  exceedingly  evanescent  action  in  contrast  to 
dimethylenimin.  The  toxic  phenomena  develop  rapidly  and  dis- 
appear equally  so  without  leaving  behind  any  permanent  injuries, 
especially  destruction  of  the  papillae.  In  contrast  to  this,  vinylamin 
is  characterized  by  a  slowly  developing  action,  which  in  small  doses- 
may  show  several  hours'  incubation  period  and  leaves  the  organism 
permanently  damaged.  I  have  compared  this  action  with  that  of 
several  other  compounds  which  I  have  studied;  thus  camphylamin,. 
which  according  to  Duden  has  the  composition 

X-NH2 
^CH 

allylamin  with  a  double  bond  (ethylene  radical): 

CH 

II 
CH 

C/' 


\ 


\H 


432  COLLECTED  STUDIES  IN   IMMUNITY. 

and  propargylamin,  which  contains  the  acetylen  group, 

C-H 


NH2 

All  of  these  substances  were  found  to  possess  the  evanescent 
general  symptoms  together  with  an  absence  of  permanent  organic 
injuries.  Hence  I  believe  that  the  chemical  avidity  of  the  double 
and  triple  combinations  is  insufficient  to  effect  substitutive  reac- 
tions with  the  protoplasm.  I  am  strengthened  in  this  view  by  the 

CH 

fact  that  prussic  acid,  which  owing  to  its  threefold  combination  ||| 

N 

can  be  classed  with  the  most  active  substances  known  to  chemistry, 
is  nevertheless  not  anchored  in  the  animal  body,  as  can  be  seen  from 
Geppert's  findings  already  referred  to. 

If  we  consider  that  substances  which  possess  double  or  triple 
bonds  are  usually  much  more  poisonous  than  the  corresponding 
saturated  combinations,1  and  if  we  bear  the  above  considerations  in 
mind,  we  shall  ascribe  this  increased  toxicity  not  to  a  combining 
capacity  but  to  the  fact  that  the  unsaturated  groups  possess  auxotoxic 
properties,  i.e.,  that  they  are  able  to  increase  the  toxicity  when  they 
enter  into  complexes  which  in  themselves  already  possess  certain 
toxic  properties. 

I  must  emphasize  the  fact  that  all  observations  thus  far  made 
&re  only  to  be  applied  to  organic  substances  foreign  to  the  body 
We  must,  however,  assume  that  all  substances  which  enter  into  the 
construction  of  the  protoplasm  are  chemically  fixed  by  the  proto- 
plasm. A  distinction  has  always  been  made  between  substances 
capable  of  assimilation,  which  serve  the  nutrition  and  enter  into  a 
permanent  combination  with  the  protoplasm,  and  substances  foreign 
to  the  body.  No  one  believes  that  quinine  and  similar  substances 
are  assimilated,  i.e.,  enter  into  the  composition  of  the  protoplasm. 
The  foodstuffs,  however,  are  bound  in  the  cell,  and  this  union  must 
be  regarded  as  a  chemical  one  The  sugar  molecule  cannot  be  ab 

1  Neurin  is  twenty  times  as  toxic  as  cholin  (trimethyiethylammonium 
hydroxide);  allylalcohol  fifty  times  more  toxic  than  propyl  alcohol;  ct  also 
Low,  Natiirliches  System  der  Giftwirkungen  1893,  page  95. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION      433 

stracted  from  the  cells  with  water;  it  must  first  be  split  off  by  means 
of  acids  in  order  to  set  it  free.  Such  a  chemical  union,  however,  just 
as  every  synthesis,  presupposes  the  presence  of  two  combining  groups 
of  maximal  chemical  affinity  which  are  fitted  to  one  another.  Those 
groups  in  the  cell  which  anchor  foodstuffs  I  term  "side-chains"  or 
"receptors;"  the  combining  group  of  the  food  molecule  the  "hap- 
tophore  group."  Hence  I  assume  that  the  living  protoplasm  pos- 
sesses a  large  number  of  such  "side-chians"  and  that  these  in  virtue 
of  their  chemical  constitution  are  able  to  anchor  the  greatest  variety 
of  foodstuffs.  In  this  way  the  cell's  metabolism  is  made  possible. 

This  view  of  the  constitution  of  the  protoplasmic  molecule  has 
made  it  possible  to  get  a  much  clearer  insight  into  the  action  of  the 
toxins  and  into  the  hitherto  mysterious  phenomenon,  the  formation 
of  antibodies.  I  assume  that  the  toxins,  just  like  the  food  mole- 
cules, possess  a  particular  haptophore  group,  which,  by  fitting  into 
the  receptor  of  the  cell,  gives  rise  to  the  poisonous  action.  Putting 
this  receptor  out  of  action  causes  a  formation  of  new  receptors  to 
replace  it,  and  these  are  finally  thrust  off  into  the  blood.  The  re- 
ceptors thus  present  in  the  blood  constitute  the  antitoxin.  This 
theory,  known  as  the  "side-chain  theory/'  has  proven  its  worth  in 
the  hands  of  numerous  investigators,  for  by  its  means  the  manifold 
reactions  of  immunity  are  all  led  back  to  the  simplest  processes  of 
cellular  life.1 

Hence  I  assume  the  presence  of  a  haptophore  group  only  in  such 
combinations  which,  like  the  foodstuffs,  enter  into  the  substance  of  the 
protoplasm,  or  which,  like  the  large  number  of  poisonous  and  non-poison- 
ous metabolic  products  of  living  cells,  effect  a  union  similar  to  that  of  the 
foodstuffs. 

The  marked  difference  between  the  two  classes  of  substances 
becomes  plainly  evident  by  the  fact  that  only  those  substances  possess- 
ing haptophore  groups  are  able  to  excite  the  production  of  antibodies 
through  immunization.  And  despite  the  most  painstaking  efforts 
neither  other  investigators  nor  I  have  ever  succeeded  in  producing 
any  appreciable  production  of  antibody  with  alkaloids,  glucosides, 
or  drugs  of  well-known  chemical  constitution. 

1  I  content  myself  here  with  these  brief  remarks  and  refer  the  reader  to 
my  more  recent  detailed  articles:  1.  On  Immunity,  etc.,  Croonian  Lecture, 
Proceedings  of  the  Royal  Soc.,  Vol.  66,  1900.  2.  Schlussbetrachtungen  zur 
Anaemic,  in  Xothnagel's  Handbuch,  Vol.  VIII,  1901.  pages  555  et  seq.  3  Die 
Schiitzstoffe  des  Blutes,  page  364  of  this  volume. 


434  COLLECTED  STUDIES  IN  IMMUNITY. 


VII. 

In  the  case  of  the  chemically  defined  poisons,  drugs,  and  dyes 
discussed  above,  incorporation  into  the  protoplasmic  molecule  does 
not,  barring  a  few  exceptions,  take  place  by  means  of  synthesis. 
Since,  however,  almost  the  greater  part  ol  all  substances  foreign  to  the 
body  exhibit  a  typical  selective  action  in  the  tissues,  it  becomes  neces- 
sary to  study  the  reasons  for  this  action.  Here  again  we  shall  do  best 
to  begin  with  a  consideration  of  the  phenomena  which  takes  place 
in  staining  reactions.  A  cotton  fibre  placed  in  a  dilution  of  picric 
acid  of  one  to  a  million  takes  up  the  dye,  becoming  intensely  stained. 
Methylene  blue  introduced  intra  vitarri  into  the  organism  is  taken 
up  by  the  nerve  endings.  In  poisoning  by  alkaloids  certain  nerve 
centres  may  react  specifically  and  alone.  All  of  these  phenomena 
are  obviously  analogous  in  their  nature.  It  seems  necessary,  therefore, 
to  discuss  briefly  the  views  held  concerning  the  nature  of  the  staining 
process.  The  purely  mechanical  conception  which  refers  it  all  to 
physical  processes,  such  as  surface  attraction  and  absorption,  can 
probably  be  discarded  for  the  staining  of  substances  in  general.  This 
leaves  only  two  other  explanations,  either  of  which  may  be  the  cor- 
rect one  for  certain  cases. 

The  first  of  these,  maintained  particularly  by  Knecht,  proceeds 
from  the  assumption  that  certain  constituents  of  the  fibre  substance 
form  with  the  dye  insoluble  salt-like  combinations  usually  termed 
laky  combinations.  This  conception  is  supported  by  the  fact  that  by 
treatment  with  alkalies  an  acid  can  be  obtained — lanuginic  acid 
derived  from  wool,  and  nucleic  acid  from  nuclear  substances — which 
possesses  the  property  of  precipitating  the  salts  of  basic  dyestuffs 
even  out  of  very  dilute  solutions.  Analogous  conditions  are  found 
to  a  great  extent  in  vital  stainings.  I  need  only  remind  the  reader 
of  the  investigations  of  Pfeiffer.  These  show  that  in  the  vital  staining 
of  plant-cells  one  can  frequently  observe  that  the  staining  is  due  to 
conspicuous  granules  of  the  almost  insoluble  tannate  of  methylene 
blue.  Naturally  in  the  higher  animals  secretion  substances  present 
in  the  cells  and  constituting  precipitants  which  form  laky  combina- 
tions can  play  a  part  in  localization. 

The  second  theory,  one  which  associates  the  staining  process 
with  the  phenomenon  of  solid  solutions,  we  owe  to  the  researches 
of  O.  N.  Witt.  This  investigator  starts  with  the  fact  that  silk  dyed 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION       435 

with  rhodamin  exhibits  a  beautiful  fluorescence.  Rhodamin  itself, 
however,  shows  fluorescence  only  when  in  solution;  when  in  the  dry 
state,  even  in  the  finest  possible  form,  it  merely  shows  a  pure  red 
color.  Because  of  this  fluorescence  Witt  assumes  that  the  dye  forms 
a  homogeneous  mixture  with  the  fibres  of  the  silk,  i.e.,  it  is  in  the 
form  of  a  solution.  Since  the  fibre,  however,  is  a  solid  substance 
this  solution  must  be  what  Yan't  HofT  terms  a  "solid  solution."  We 
know  that  the  same  dye  often  produces  different  tints  in  various  kinds 
of  fibres.  This  is  analogous  to  the  fact  that  the  same  substance 
often  dissolves  in  different  solvents  in  entirely  different  tints,  as  is 
the  case,  for  example,  with  iodine.  Witt  therefore  believes  that 
the  process  of  staining  proceeds  exactly  the  same  as  the  distribution 
of  a  substance  in  two  different  solvents.  Thus,  if  we  dissolve  anilin 
in  water,  we  find  that  we  can  shake  all  the  anilin  out  tvith  ether y 
because  the  solvent  power  of  the  ether  is  greater  than  that  of  water.  In 
the  staining  process  such  a  vast  difference  in  solvent  power  shows 
itself  by  the  fact  that  the  materials  introduced  entirely  exhaust  the 
staining-bath.  If,  however,  the  difference  in  solvent  powers  is  less 
than  this,  e.g.  in  the  combination  water,  ether  and  resorcin,  we  shall 
find  that  the  resorcin  is  distributed  between  both  fluids  in  accordance 
with  a  law  of  distribution  which  can  be  figured  out  mathematically 
for  every  case.  In  dyeing  this  type  corresponds  to  the  dyes  which  are 
said  to  "take"  poorly.  In  these  the  staining-bath  does  not  become 
exhausted  under  ordinary  conditions.  Exhaustion  can  be  effected 
only  through  the  addition  of  certain  substances  which  limit  solution 
(salt  dyes,  etc.). 

In  the  introductory  chapter  I  have  already  mentioned  that  all 
neurotropic  and  lipotropic  substances  lose  the  property  to  stain  brain 
substance  and  fat  by  the  introduction  of  the  sulfonic  acid  radical. 
If  these  substances  are  examined  in  a  test-tube  it  is  found  that  this 
substitution  has  caused  them  to  lose  also  the  solubility  in  ether  or 
in  fats.  Thus,  although  flavanilin  is  easily  taken  up  by  ether  from 
an  alkaline  solution,  not  a  trace  of  flavanilinsulfonic  acid  is  taken  up. 
Another  interesting  case  may  be  mentioned,  one  which  concerns 
staining  with  neutral  red.  This  has  the  following  formula: 


NH2  N  N(CH3)2 


436  COLLECTED  STUDIES  IN   IMMUNITY 

This  substance  has  the  property  of  staining  the  granules  of  cells 
most  intensely,  and  the  same  holds  true  of  a  number  of  derivatives, 
e.g.  violet  dimethyl  neutral  red,  in  which  the  two  hydrogens  of  the 
second  amido  group  are  replaced  by  two  methyl  groups;  further, 
also,  the  golden-red  diamidophenazin: 


NH2  N  N(CH3)2 


In  contrast  to  this,  however,  the  combination  in  which  one  of  the 
central  amin  radicals  contains  an  ethyl  group  which  gives  to  the 
group  the  character  of  an  ammonium  base,  is  absolutely  unable  to 
effect  the  staining.  All  phenazin  derivatives  which  stain  granules  can 
be  completely  shaken  out  of  weak  alkaline  solutions  by  means  of 
ether,  whereas  not  even  a  trace  of  the  ammonium  base  belonging 
to  the  safranin  series  is  thus  taken  up  by  the  ether 

A  very  intimate  connection,  however,  exists  between  solubility  in 
the  test-tube  and  ability  to  be  absorbed  in  the  organism,  a  connection 
which  I  observed  as  long  as  fifteen  years  ago.  Hence  we  must  assume 
that  certain  fat-like  substances  of  the  nervous  system  as  well  as  the 
fat  of  fat  cells  possess  a  high  solvent  power  by  means  of  which  these 
substances  are  anchored  or  stored  up  in  the  tissue  in  question,  just 
as  the  alkaloids  are  taken  up  by  the  ether  in  the  Stas-Otto  pro- 
cedure.1 

If  we  bear  in  mind  not  only  the  extraordinary  multiplicity  of 
Substances  foreign  to  the  body,  but  also  the  varying  chemistry  of 
the  tissues  which  make  up  the  organism,  we  shall  not  expect  that 
a  single  principle  can  be  rigidly  applied  to  the  phenomenon  of 

1  This  behavior  has  been  studied  especially  by  Overton.  He  terms  the 
substances  of  the  brain  which  serve  as  extracting  agents  "hpoids."  Chief 
among  these  are  cholesterin  and  lecithin  Among  the  alkaloids  Overton  dis. 
tinguishes  feebly  basic  and  more  strongly  basic  substances.  The  former  can  be 
shaken  out — for  example,  the  indifferent  narcotics;  whereas  the  more  strongly 
basic  unite  with  constituents  of  the  cell  to  form  salt-like  combinations  which 
are  very  easily  dissociated.  According  to  Overtoil's  conception  therefore 
Knecht's  explanation  would  apply  at  one  time  and  Witt's  at  another 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION.      437 

selective  action.  For  a  large  number  of  substances  which  localize 
in  fat  or  fat-like  bodies  during  life,  it  will  probably  be  difficult  to  prove 
whether  a  pure  shaking-out  process  occurs  or  a  formation  of  but 
slightly  soluble  salts. 

Furthermore,  both  processes  may  occur  toegther,  as  Knecht  as- 
sumes in  dyeing,  the  lake-forming  components  being  contained  in 
the  tissues  in  the  intimate  molecular  mixture  characteristic  of  solid 
solutions.  In  that  case  the  resulting  selective  action  will  be  due 
to  a  combination  of  salt  formation  and  solid  solution.  In  many 
instances,  however,  it  will  be  extremely  difficult  to  decide  whether 
one  is  dealing  with  solid  solution  or  salt  or  double-salt  formation, 
especially  since  chemistry  often  finds  it  impossible  to  decide  this 
question  in  the  case  of  pure  bodies.  This  is  seen,  for  example,  in  the 
study  of  mixed  crystals  which  are  looked  upon  mostly  as  crystalline 
solutions.1 

In  any  case  we  see  that  even  without  the  intervention  of  a  chemic- 
synthetic  union  the  conditions  necessary  for  a  selective  storage  of  a 
substance  in  the  organism  are  present  and  are  sufficient  both  in 
extent  and  in  variety.2  That  these  conditions  in  the  case  of  the 
salt-like  combinations  are  essentially  chemical  in  nature  is  self-evident; 
in  the  case  of  the  solid  solution  the  enormous  mass  of  evidence  which 
I  have  merely  touched  makes  this  extremely  probable.  If  we  regard 
the  principles  governing  distribution  in  the  organism  from  these 
standpoints  we  shall  no  longer  be  surprised  that  in  the  localization 

1  If  two  combinations  of  somewhat  similar  chemical  constitution  (for  ex- 
ample, benzole  and  pyridin;  stilben,  benzylidenanilin,  and  azobenzole;  fluoren 
and  diphenylenoxid)  form  mixed  crystals  with  each  other,  one  can  readily 
comprehend  this  in  view  of  their  close  chemical  relationship,  and  can  ascribe 
it  to  "isomorphogenous"  groups.  Frequently,  however,  substances  crystallize 
together  which  exhibit  the  greatest  divergence  in  the  configuration  of  their 
molecules,  as,  for  example,  phenol  and  urea,  chloroform  and  salicylid,  triphenyl- 
methan  and  benzol.  The  crystalline  fiery-colored  combinations  which  picric 
acid  is  able  to  effect  with  a  large  number  of  hydrocarbons  are  especially  im- 
portant. Certain  investigations  concerning  the  basic  properties  of  oxygen 
(Baeyer)  and  of  carbon  (Kehrman  and  Baeyer)  seem  to  show  that  such  crys- 
tallizations, as,  for  instance,  of  ferrohydrocyanic  acid  with  ether,  etc.,  are  anal- 
ogous of  salt  formation. 

2 1  must  here  refer  the  reader  to  the  extremely  interesting  investigations 
of  Spiro  (Uber  physikalische  und  physiologische  Selection,  Habilitationsschrift, 
Strassburg  1897).  In  these,  although  starting  from  entirely  different  stand- 
points *he  author  reaches  many  of  the  views  held  by  me.  At  the  time  of  my 
address  I  was  unaware  of  this  study,  as  it  is  not  to  be  had  in  the  bookshops. 


438  COLLECTED   STUDIES  IN   IMMUNITY 

of  substances  foreign  to  the  body  synthetic  processes  play  practically 
no  role  whatever.  If  we  take  methylene  blue  as  an  example,  we  see 
at  once  that  we  can  easily  find  a  large  number  of  different  fluids 
which  are  able  to  shake  it  out.  On  the  other  hand,  we  know  of  a 
large  number  of  acids,  like  picric  acid,  phosphomolybdic  acid,  hyper- 
sulphuric  acid,  which  are  able  to  precipitate  the  methylene  blue  in 
insoluble  form  even  out  of  very  dilute  solutions.  This  dyestuff,  how- 
ever, is  practically  useless  for  synthetic  processes;  all  the  efforts  of 
the  chemists  to  introduce  other  groups  into  the  completed  molecules 
(with  one  exception,  nitro-methylene  blue)  have  absolutely  failed. 
When  we  stop  to  consider  that  in  such  chemical  procedures  the 
strongest  possible  agents  can  be  used,  sulphuric  acid,  high  tempera- 
tures, etc.,  we  shall  at  once  see  that  methylene  blue  cannot  at  all  be 
synthetically  bound  in  the  organism.  The  extensive  distribution  of 
methylene  blue,  however,  is  very  easily  explained  by  the  plentiful 
opportunities  offered  for  localization. 

Synthetic  processes,  such  as  occur  in  the  absorption  of  foodstuffs, 
in  assimilation,  and  in  the  growth  of  living  matter,  are  connected 
with  the  existence  of  certain  chemical  groups,  the  "receptors." 
These  receptors  are  able  to  synthesize  with  fitting  haptophore  groups 
of  the  foodstuffs  or  of  the  toxins,  the  two  groups  fitting  specifically 
to  each  other  (like  lock  and  key:  E.Fischer).  The  eagerness  with 
which  the  living  protoplasm  lays  hold  of  the  foodstuff  which  it  re- 
quires is  in  marked  contrast  to  the  manner  in  which  it  resists  taking 
up  substances  foreign  to  itself.  This  was  observed  even  in  the  begin- 
ning of  histology,  for  at  that  time  it  was  regarded  as  an  axiom  that 
living  cells  could  not  possibly  be  stained.  Gerlach,  for  example, 
had  shown  that  an  amoeba  does  not  take  up  any  coloring  matter  from 
a  solution  of  carmine,  whereas  it  stains  immediately  when  it  is  dead. 
Since  then,  to  be  sure;  largely  through  my  efforts,  we  have  come  to 
know  a  number  of  important  vital  stains  (neutral  red,  methylene 
blue,  brilliant  cresyl  blue),  but  closer  analysis  of  these  phenomena 
have  shown  that  that  which  can  be  demonstrated  in  the  living  cell 
by  the  various  dyes  is  not  the  functionating  protoplasm  but  its 
lifeless  (paraplastic)  surrounding  medium  and  the  granules,  etc., 
present  therein.  In  this  point  I  agree  entirely  with  Galeotti. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION       439 


VIII. 

What  practical  conclusions  can  be  drawn  from  these  considera- 
tions? We  see  that  drugs,  such  as  the  majority  of  narcotics— in 
fact  the  large  number  of  neurotropic  and  lipotropic  substances — be- 
come localized  through  a  shaking-out  process.  It  follows  from  what 
has  already  been  said  that  only  such  substances  can  be  anchored  at  any 
particular  part  of  the  organism  which  fit  into  the  molecule  of  the 
recipient  combination  as  a  piece  of  mosaic  fits  into  a  certain  pat- 
tern. Such  configurations,  however,  are  not  confined  to  a  single 
substance,  but  usually  include  a  large  group  of  related  substances. 
In  this  connection  the  investigations  which  Einhorn l  and  I  made 
concerning  the  action  of  cocaine  are  most  important. 

Cocaine  is  a  derivative  of  ecgonin,  whose  molecule  contains  two 
groups  differing  in  function:  a  hydroxyl  group,  which  combines  with 
acid  radicals,  and  a  carboxyl  group,  which  forms  esters  with  alcohol 
radicals.  All  derivatives  of  ecgonin  in  which  both  groups  are  thus 
occupied  represent  bodies  of  the  cocaine  series.  Thus  in  the  cocaine 
ordinarily  used  in  medicine  the  acid  radical  is  that  of  benzoic  acid, 
the  ester  former  is  a  methyl  group.  By  means  of  the  methods  of 
modern  chemistry  it  has  been  possible  to  introduce  the  greatest  variety 
of  radicals  into  ecgonin,  leading  to  the  formation  of  a  large  number 
of  homologous  substances.  It  was  soon  found  that  the  substitution 
of  other  alcohol  radicals,  such  as  ethyl,  propyl,  etc.,  for  the  methyl 
radical  did  not  cause  the  least  change  in  the  physiological  effects 
of  the  cocaine,  as  Falk  proved.  On  the  other  hand,  the  acid  radical 
is  of  prime  importance  for  the  anesthetic  action  of  the  cocaine.  Pouls- 
son,  Liebreich,  and  myself  studied  the  various  cocaines  with  other 
acid  radicals  (cinnamyl  cocaine,  phenacetyl  cocaine,  valeryl  cocaine, 
phthalyl  cocaine)  and  found  only  one,  the  phenylacetic  acid  derivative, 
which  possessed  even  feeble  anaesthetic  properties.  As  a  result  of 
these  toxicological  experiences  one  could  have  assumed  that  this 
benzoyl  cocaine  was  in  every  way  unlike  all  other  acid  derivatives. 
But  this  is  not  the  case,  for  I  was  able  to  show  that  so  far  as  another 
toxic  action  is  concerned  all  of  the  various  cocaines  show  the  same 

1  Einhorn  is  one  of  the  best  authorities  on  alkaloids  known  to  me.  The 
studies  referred  to,  appear  in  the  Deutsche  med.  Wochensch.  1890,  No.  32,  and  in 
Berichte  der  deutschen  chem.  Gesellschaft  1894,  Vol.  27,  page  1870. 


440  COLLECTED  STUDIES  IN   IMMUNITY. 

behavior,  namely,  in  mice  they  all  produce  a  peculiar  foam-like  degen- 
eration of  the  liver-cells  which  I  have  observed  only  in  substances 
belonging  to  this  series.  From  this  it  follows  that  all  bodies  of  the 
cocaine  series  are  alike  so  far  as  the  liver  is  concerned.  Considering 
that  the  substances  which  precipitate  and  dissolve  these  bodies  are 
the  same  and  that  the  liver  findings  are  identical,  we  may  perhaps 
assume  that  all  cocaines  are  taken  up  by  the  liver  in  the  same  way 
and  therefore  probably  also  by  the  other  parenchyma.  And  since 
the  benzoyl  derivative  is  the  only  one  which  possesses  anaesthetic 
action  we  shall  have  to  assume  that  the  rest  of  the  molecule  is  only 
the  carrier  which  brings  the  benzoic  acid  radical  to  the  proper  place. 
(The  anaesthesiophore  character  of  this  group  had  already  been  made 
very  probable  by  the  earlier  investgations  of  Filehne.)  Let  us  go 
back  to  our  illustration  of  the  mosaic  in  order  to  get  this  idea  clearly 
before  us.  In  order  for  a  piece  to  help  complete  a  given  figure  it  is 
first  necessary  that  it  possess  a  particular  form,  but  in  order  that  the 
pattern  be  really  completed  the  piece  must  also  possess  certain  material 
properties,  such  as  hardness,  color,  lustre,  etc.  It  will  be  one  of  the 
problems  of  the  future  to  extend  our  knowledge  concerning  the  active 
toxophore  groups. 

The  first  fundamental  experiments  in  this  direction  were  made 
by  Ladenburg,  who  showed  that  the  two  substances  obtained  on 
splitting  atropin,  namely,  tropin  and  tropic  acid,  could  readily  be 
recombined  and  the  atropin  molecule  thus  be  reconstructed.  As  a 
result  of  this  demonstration  that  atropin  represents  an  acid  ester 
of  tropin  it  was  possible  to  produce  a  number  of  homologous  combina- 
tions, Ladenburg's  "tropeins,"  e.g.,  benzyltropein,  salicyltropein, 
phenylglycoltropein  (homa tropin).  A  comparative  study  of  the  these 
substances  showed  that  for  mydriatic  purposes  aromatic  oxyacids 
were  the  most  favorable — and  especially  those  in  which  the  hydroxyl 
is  in  aliphatic  combination,  as  in  tropic  acid  and  phenylglycolic  acid. 

In  cocaine,  Einhorn  and  I  attempted  to  determine  the  function 
of  the  benzoyl  group  by  introducing  various  side-chains.  It  was 
found  that  the  introduction  of  a  nitro  group  in  the  meta  position  had 
a  marked  influence  on  the  anaesthetizing  property  of  cocaine  without 
preventing  the  injurious  action  on  parenchyma  described  above.  The 
introduction  of  a  hydroxyl  group  in  the  same  place  acted  still  more 
strongly  in  this  direction,  for  the  anaesthetizing  property  had  dis- 
appeared, the  toxic  action  on  the  liver  decreased.  Meta-amido  cocaine 
was  entirely  inert. 


CHEMICAL  CONSTITUTION  AND  PHARMACOLOGICAL  ACTION       441 

What  was  extremely  interesting  was  the  fact  that  by  the  intro- 
duction of  suitable  radicals  into  this  inert  amido  cocaine  the  alka- 
loidal  action  could  be  restored.  Thus  when  acetyl  and  benzoyl 
groups  are  introduced  into  amido  cocaine,  cocaines  are  formed  which, 
although  they  are  not  anaesthetic,  again  possess  this  property  of 
acting  on  the  liver.  It  is  especially  interesting,  however,  that  the 
cocaine  urethane  obtained  by  the  action  of  chlorcarbonic  acid  on 
amido  cocaine  again  acts  anaesthetically,  in  fact  much  more  so  than 
the  original  cocaine.  That  is  to  say,  if  we  nitrify  cocaine,  reduce  it 
to  amido  cocaine,  and  finally  condense  it  to  a  urethane,  we  find  that 
the  anaesthesiophore  group  is  first  diminished  in  power,  then  its 
action  is  entirely  lost,  and  finally  heightened.  We  already  know  the 
function  of  the  toxopho:  e  group  in  a  number  of  alkaloids,  in  atropin 
for  a  single  group,  in  strychnine  for  two.  If  only  we  had  a  deeper 
insight  into  this  function  we  might  hope  by  means  of  substitutive 
action  on  the  toxophore  groups  (such  as  Einhorn  and  I  have  car- 
ried out  on  the  benzoic  acid  radical  of  cocaine)  to  modify  the  action 
of  the  alkaloids  to  suit  our  purpose. 

In  the  synthetic  field  of  pharmacology,  however,  a  knowledge  of 
the  groupings  on  which  the  selective  distribution  in  the  organs  depends 
would  appear  to  be  far  more  important.  In  the  case  of  foodstuffs 
and  toxins  I  assume  that  the  union  is  effected  by  a  single  definite 
group,  the  "haptophore"  group.  Substances  foreign  to  the  body, 
as  already  explained,  lack  such  a  single  group  and  the  laws  of  dis- 
tribution in  the  organism  are  dependent  on  the  combined  action  of 
the  separate  components.  In  their  distribution,  therefore,  the  entire 
constitution  of  the  substance  is  the  deciding  factor.  This  we  have  seen 
to  be  true  with  substances  belonging  to  one  group.  Within  this 
group  type,  as  we  have  described  it  in  detail  with  the  cocaine  series, 
modifications  of  the  separate  components  can  then  be  made  within 
wide  limits.  Starting  from  this  point  of  view  we  obtain  a  new  method 
of  synthetic-chemical  pharmacology.  If  one  is  desirous  of  studying 
organ  therapy  in  this  sense  it  will  be  necessary  first  to  hunt  up 
bodies  which  possess  a  particular  affinity  for  a  certain  organ. 
Having  found  such  bodies  one  can  then  use  them,  so  to  speak,  as  a 
carrier  by  which  to  bring  therapeutically  active  groups  to  the  organ 
in  question.  It  is  self-evident  that  in  the  selection  of  these  groups 
one  is  bound  by  definite  limits;  so  also  is  the  fact  that  all  substituting 
groups  which  themselves  influence  the  distributive  character  (e.g. 
acid  radicals)  must  be  avoided.  All  these  are  problems  which  ex- 


442  COLLECTED  STUDIES  IN  IMMUNITY. 

tend  far  beyond  the  powers  of  single  individuals  and  make  it 
desirable  -that  chemists  and  pharmacologists  work  together  in  some 
definite  plan.  That  is  one  reason  why  I  have  gone  into  such  detail 
concerning  my  views  on  the  connection  between  constitution,  dis- 
tribution in  the  organs,  and  pharmacological  action.  1  shall  indeed  be 
happy  if  these  views,  the  gradual  development  of  ten  years  of  study, 
will  advance  the  study  of  pharmacology. 

TRANSLATOR'S  NOTE. — See  also  the  recently  published  study  by  Bechhold 
and  Ehrlich  on  the  relation  of  chemical  constitution  to  disinfecting  power. 
<Zeitschrift  fiir  physiol.  Chemie,  Vol.  XL VII,  Nos.  2  and  3,  1906.) 


XXXV.   A  STUDY  OF  THE  SUBSTANCES  WHICH 
ACTIVATE  COBRA  VENOM.1 


BY 

Dr.  PRESTOX  KYES,  Dr    HANS  SACHS, 

Associate    in    Anatomy,    University   of  Assistant  at  the  Royal  Institute 
Chicago,   Fellow  of  the  Rockefeller  for  Experimental  Therapy, 

Institute  for  Medical  Research.  Frankfurt-on-Main. 

I.  Concerning  the  Activation  of  Cobra  Venom  by  Means  of 
Complements. 

Ix  a  previous  study2  one  of  us  has  shown  that  cobra  venom  is 
activated  not  only  by  certain  active  sera  but  also  by  lecithin  and 
certain  complement-like  substances  of  the  red  blood-cells  called 
"endocomplements."  This,  of  course,  harmonizes  with  the  ambo- 
ceptor  nature  of  the  poison  which  had  been  demonstrated  by  Flexner 
and  Xoguchi.3  In  view  of  the  wide  distribution  of  lecithin  in  the 
organs  and  tissues  it  seemed  advisable  to  penetrate  deeper  into  the 
mechanism  of  cobra-venom  haemolysis,  especially  in  order  to  deter- 
mine if  the  assumption  of  complements  and  endocomplements  is 
not  superfluous  and  the  presence  of  lecithin  in  the  red  blood-cells 
and  serum  sufficient  to  explain  the  complement  action.  It  is  true 
that  certain  sera  which  activate  cobra  venom  lose  this  property  when 
they  are  heated  to  56°  C.  for  half  an  hour,  and  the  endocomplements 
produced  by  dissolving  the  red  blood-cells  in  water  are  inactivated 
by  heating  to  62°  C.  Considering  the  great  ease  with  which  lecithin 
combines  with  albuminous  bodies,  etc;,  it  was  possible  that  the 
thermolability  of  the  activating  factors  was  simulated  by  a  com- 
bination of  the  lecithin  with  other  substances.  An  important  fact 
which  speaks  strongly  against  this  view,  however,  is  one  first  brought, 

1  Reprint  from  the  Berlin  klin.  Wochensch.  1903,  Xos.  2  and  4. 

2  P.  Kyes.     See  page  291. 

3  Flexner  and  Xoguchi,  Snake  Venom  in  Relation  to  Haemolysis,  Bacteri- 
olysis, and  Toxicity,  Journ  of  Exp.  Medicine,  Vol.  VI,  No.  3,  1902. 

443 


444  COLLECTED  STUDIES  IN  IMMUNITY 

out  by  Calmette,1  namely,  that  almost  all  sera  after  being  heated  to 
65°  C.  and  higher  usually  show  even  an  increased  activating  power. 
This  we  could  explain  only  by  ascribing  it  to  the  lecithin  set  free 
through  heating  (Kyes,  1.  c.).  It  thus  appeared  that  heating  was  more 
likely  to  effect  a  splitting  off  than  a  combination  of  the  lecithin. 

Our  further  studies  have  shown,  however,  that  this  view  is  not 
correct  in  all  cases.2 

To  begin,  we  examined  the  complementing  properties  of  serum, 
choosing  for  our  analysis  the  combination  ox  blood  H-  cobra  venom  + 
guinea-pig  serum.  The  activating  property  of  the  fresh  guinea-pig 
serum  was  destroyed  by  half  an  hour's  heating  to  56°  C.,  and  hence 
was  apparently  not  due  to  the  presence  of  lecithin,  but  to  some  other 
complement-like  substance.  Subsequent  investigations  have  con- 
firmed us  in  this  opinion.  The  general  course  of  the  haemolysis 
through  snake  venom  is  markedly  different  when  lecithin  or  serum 
is  used  for  complementing.  Lecithin  effects  rapid  solution;  with 
large  amounts  of  cobra  venom  this  is  almost  instantaneous.  When. 
serum  is  used  as  complement  a  longer  or  shorter  period  of  incubation 
is  observed,  such  as  we  are  accustomed  to  see  with  the  haemolytic 
sera.  Furthermore,  haemolysis  with  cobra  venom  +  lecithin  occurs 
even  at  0°  C.,  whereas  the  action  of  cobra  venom  +  serum  as  comple- 
ment requires  a  greater  degree  of  heat. 

That  the  activating  substance  of  the  serum  belongs  to  the  class 
of  complements  was  further  demonstrated  by  the  fact  that  it  was 
destroyed  by  digestion  with  papain.  Following  the  method  of  Ehrlich 
and  Sachs,3  in  order  to  digest  the  complement,  5  cc.  guinea-pig  serum 
were  mixed  with  1  cc.  10%  solution  of  papain,  digested  for  1J  hours 
and  then  centrifuged.  The  decanted  fluid  was  used  to  activate  the 
cobra  venom.  Table  I  shows  that  this  property  was  almost  com- 
pletely lost. 

The  serum  treated  with  papain  had  thus  almost  completely  lost 
its  activating  property,  whereas  a  solution  of  lecithin  similarly  treated 
preserved  its  activating  property  unchanged.  (See  Table  II.) 


1  Calmette,  Sur  1'action   hemolytique  du  venin  de  cobra,  Compt.   rend,   de 
1' Academic  des  Sciences,  T.  134,  No.  24,  1902. 

*  We  are  much  indebted  to  Drs.  Lamb  and  Greig  for  cobra  venom  kindly 
placed  at  our  disposal. 

*  Ehrlich  and  Sachs,  The  Plurality  of  Complements  in  Serum,  Berl.  klin 
Wochenschr.  1902,  Nos  14  and  15. 


SUBSTANCES  WHICH   ACTIVATE   COBRA   VENOM. 
TABLE   I 


445 


Amount  of 
Serum. 

cc. 

1  cc  5%  Ox  Blood  -l-O  02  cc.  1%  Cobra 
Venom  +  Guinea  pig  Serum. 

(a)  Normal. 

(6)  After  Previous 
Treatment  with 
Papain. 

Amount  of  Haemolysis. 

0.5 

0.35 
0.25 
0.15 
0.1 
0.075 

complete 

almost  complete 
strong 

moderate 

almost  0 
"      0 

•'      0 
"      0 
0 
0 

TABLE  II. 


Amount  of 
Lecithin 
Solution. 

cc. 

1  cc   5%  Ox  Blood  +0.02  cc    1%  Cobra 
Venom  +  0.025%  Lecithin. 

(a)   Native 

(6)  After  Previous 
Treatment  with 
Papain. 

Amount  of  Haemolysis 

0.25 
0.15 

complete 

complete 

0.1 

(  t 

<  « 

0.075 

trace 

trace 

0.05 

0 

0 

In  like  manner  the  complementing  property  of  the  serum  is  de- 
stroyed by  appropriate  digestion  with  hydrochloric  acid  and  with 
soda  lye,  in  which  again  the  serum  differs  from  lecithin. 

We  felt  that  the  discovery  of  agents  which  would  exert  an  inhibit- 
ing effect  in  the  hsemolytic  action  of  only  one  of  the  two  factors  (either 
on  that  of  the  serum  or  of  the  lecithin)  would  be  most  valuable  for 
a  positive  differentiation  of  serum  complement  and  lecithin.  We 
therefore  next  immunized  rabbits  and  chickens  with  guinea-pig  serum, 
seeking  in  that  way  to  produce  anticomplements.  By  the  natural 
production  of  an  antibody  we  could  thus  prove  the  complement 
nature  of  the  serum  activator.  These  experiments,  however,  en- 
countered certain  difficulties,  for,  as  we  have  already  mentioned 


446 


COLLECTED   STUDIES   IN   IMMUNITY 


normal  sera  exert  a  considerable  inhibition  on  cobra  venom  haemolysis 
when  serum  is  used  as  complement,  and  to  a  still  greater  degree  when 
lecithin  is  used.  We  observed  no  essential  increase  in  the  protective 
action  in  the  animals  treated  with  guinea-pig  serum.  We  therefore 
next  sought  to  distinguish  anticomplement  and  antilecithin  action 
in  normal  serum. 

For  this  purpose  guinea-pig  serum  itself  seemed  best  suited; 
inactivated  by  half  an  hour's  heating  to  56°  C.  it  exerts  a  marked 
inhibitory  action  on  cobra  venom  +  lecithin  ha?molysis.  This  fact 
by  itself,  however,  in  no  way  argues  against  the  identity  of  lecithin 
and  the  complementing  substance  of  active  guinea-pig  serum.  One 
could  assume,  for  instance,  that,  on  heating,  a  substance  is  formed 
which  is  capable  of  combining  with  the  lecithin.  In  that  case  if  an 
excess  of  the  substance  were  formed,  this  would  be  capable  of  com- 
bining with  lecithin  subsequently  added.  This  would  explain  the 
apparently  paradoxical  phenomenon  that  the  same  serum  when  fresh 
exhibits  activating  properties,  but  when  heated  to  56°  C.  is  able  to 
bind  lecithin. 

We  therefore  investigated  the  property  of  active  fresh  guinea- 
pig  serum  to  inhibit  the  action  of  lecithin  and  hoped  that  this  prop- 
erty would  still  be  manifested  in  dilutions  in  which  the  serum  was 
no  longer  able  to  exert  any  activating  influence  on  cobra  venom. 
As  a  matter  of  fact  we  succeded  in  proving  that  guinea-pig  serum 
still  exerts  an  inhibiting  influence  on  the  lecithin,  even  in  very  small 
amounts  which  no  longer  activate.  This  is  shown  by  the  example 
in  Table  III. 

TABLE  III. 

1  cc.  5%  Ox  BLOOD +0.001  cc.  1%  COBRA  VENOM +  0.075  cc.  0.025% 

LECITHIN. 


Amounts  of  Guinea 
pig  Serum  Added, 
cc. 

Haemolysis. 

0.5 
0.25 
0.1 
0.05 
0.025 
0.01 
0 

complete 
strong 
trace 
0 
0 
trace 
complete 

The  lecithin  and  guinea-pig  serum  are  digested  for  half  an  hour  previous  to 
adding  the  ox  blood  and  cobra  venom. 


SUBSTANCES  WHICH   ACTIVATE  COBRA  VENOM.  447 

Under  these  circumstances,  of  course,  we  can  no  longer  regard 
the  activating  factor  of  guinea-pig  serum  and  lecithin  as  being 
identical.  If  lecithin  and  serum  complement  were  identical,  the 
antilecithin  should  act  also  against  the  serum  complement.  In 
guinea-pig  serum,  however,  as  is  shown  by  its  activating  power,  an 
excess  over  any  such  inhibiting  substances  is  surely  present  and  this 
excess,  of  course,  persists  even  in  quantities  of  the  serum  so  small 
as  no  longer  to  lead  to  haemolysis.  A  serum  protection  can  there- 
fore be  exerted  only  against  substances  which  are  different  from  the 
activating  substance  of  the  serum. 

Further  confirmation  of  this  difference  was  afforded  by  the  fact 
that  we  succeeded  in  demonstrating  the  existence  of  antilecithin 
and  anticomplement  components  in  normal  rabbit  serum  inactivated 
by  heating  to  56°  C.  By  the  addition  of  lecithin  we  completely 
neutralized  the  components  which  inhibit  lecithin.1  In  fact  we  added 
so  much  that  there  was  a  slight  excess  of  free  lecithin.  Although  this 
mixture  in  large  quantities  was  itself  activating,  in  smaller  quantities 
it  was  able  to  markedly  inhibit  haemolysis  with  cobra  venom  +  guinea-pig 
serum.  The  anticomplement  component  of  the  rabbit  serum  had 
been  unaffected  by  the  addition  of  lecithin,  as  can  be  seen  from  the 
following  experiment: 

20  cc.  rabbit  serum  are  mixed  with  180  cc.  absolute  alcohol,  the   resulting 
precipitate  rapidly  filtered,  pressed  out,  and  dissolved  in  20  cc.  salt   solution 
This  solution  protects  against  cobra  venom  haemolysis  not  only  with    lecithin 
activation  but  also  with  that  of  guinea-pig  serum. 

4cc.  of  the  inhibiting  solution  are  digested  for  three-quarters  of  an  hour  with 
2  cc.  of  a  0.17%  lecithin  solution.  In  large  amounts  this  mixture,  through  an 
excess  of  lecithin,  activates  cobra  venom;  in  small  amounts  it  inhibits  the 
activation  with  guinea-pig  serum.  (See  Table  IV.) 

Besides  this  we  have  discovered  that  cholesterin  markedly 
inhibits,  or  even  entirely  prevents,  the  cobra-venom  haBmolysis 
brought  about  by  lecithin.  We  shall  return  to  this  point  later.  In 
contrast  to  the  behavior  of  the  lecithin  we  find  that  the  serum  com- 
plement is  practically  unaffected  by  cholesterin,  for  only  a  very 
slight  inhibition  is  observed  even  with  large  amounts  of  choles- 
terin, a  phenomenon  which  may  be  due  to  absorption.  Such  an 
experiment  is  reproduced  in  Table  V.  The  solution  of  cholesterin 

1  In  order  to  exclude  the  activating  action  of  rabbit  serum,  which  is  due  to 
available  lecithin,  it  is  necessary  to  work  with  the  alcoholic  precipitate  obtained 
from  rabbit  serum  This  contains  the  inhibiting  substances. 


448 


COLLECTED   STUDIES  IN   IMMUNITY 


was  made  by  mixing  1  cc.  of  a  hot,  saturated  solution  of  cholesterin 
in  methyl  alcohol  with  9  cc.  hot  0.85%  salt  solution.  This  homo- 
geneous suspension  of  cholesterin  served  as  stock  solution  for  the 
experiments. 

TABLE  IV 

0.25  cc.  GUINEA-PIG  SERUM   AND  THE    INHIBITING  SOLUTION  ARE  DIGESTED 

AT  37°  C.   FOR   THREE-QUARTERS    OF    AN  HOUR.     THEREUPON   THE  Ox 

BLOOD +0.01  1%  COBRA  VENOM  ARE  ADDED. 


Haemolysis  in  the  Presence  of 

Amounts  of 

the  Inhibiting 

Solution, 
cc. 

A.  Native  Solution 
of  the  Precipitate. 

B.  Solution  of  the 
Precipitate  + 
Lecithin. 

1.0 

faint  trace 

complete 

0.5 

trace 

almost  complete 

0.25 

little 

moderate 

0.15 

<  < 

little 

0.1 

moderate 

moderate 

0.05 

4  ' 

*  * 

0.025 

strong 

strong 

0.01 

i  ( 

almost  complete 

0 

complete 

complete 

TABLE  V. 


Ox  Blood+0.01  cc.  1%  Cobra  Venom 

Cholesterin 
Solution. 

Activated  with  the  Complete 
Solvent  Dose  of 

cc. 

(a)  Guinea-pig 
Serum. 

(6)  Lecithin. 

0.5 

moderate 

0 

0.25 

«« 

0 

0.1 

1  ' 

0 

0.05 

strong 

0 

0.025 

complete 

0 

0.01 

0 

0.005 

(  ( 

0 

0.0025 

complete 

These  various  experiments  lead  us  to  believe  that  serum  comple- 
ment and  lecithin  are  two  entirely  distinct  substances.  On  the  other 
hand  the  complementing  property  of  the  serum  for  cobra  venom 
corresponds  so  well  with  the  other  complement  functions  of  the 
sera  that  no  reason  at  present  exists  for  undertaking  a  separation. 
In  conformity  with  this  correspondence  we  find  that  the  activating 


SUBSTANCES  WHICH   ACTIVATE  COBRA  VENOM.  449 

substance  is  absorbed  by  yeast.  In  the  same  connection  we  may 
perhaps  mention  that  when  fresh  guinea-pig  serum  is  shaken  with 
ether  it  loses  not  only  the  other  complementing  functions  but  also 
that  for  cobra  poison.  If  guinea-pig  serum  which  has  been  heated 
to  100°  C.  (and  which  therefore  owes  its  activating  property  to  the 
lecithin  liberated  through  heat)  is  treated  with  ether  in  exactly  the 
same  manner,  the  complementing  function  remains  unchanged. 


II.  The  Lecithin  Content  of  the  Stromata  and  the  Activation  of  Cobra 
Venom  Dependent  Thereon. 

In  the  investigation  of  the  substances  in  the  red  blood-cell  termed 
endocomplements  we  were  at  first  led  into  error  by  the  employ- 
ment of  just  this  method  of  differentiation  dependent  on  the  de- 
structibility  of  complements  by  means  of  ether. 

For  these  experiments  we  used  the  combination  ox  blood  f cobra 
venom  +  solution  of  guinea-pig  blood.  The  latter  was  obtained  by 
dissolving  sedimented  guinea-pig  blood  in  distilled  water.  The  solu- 
tion was  made  up  to  three  times  the  original  volume  of  blood,  where- 
upon NaCl  was  added  until  the  solution  contained  0.85%.  If  such 
a  solution  is  shaken  with  highly  purified  ether  (1  volume  blood 
solution -I- 10  volumes  ether)  and  a  sample  of  the  solution  (separated 
by  means  of  a  separating-funnel)  is  tested  it  will  be  found  that  this 
has  lost  its  power  to  activate  cobra  venom.  The  ethereal  residue 
taken  up  in  salt  solution  also  exhibited  no  complementing  properties, 
so  that  it  appeared  as  though  the  substance  termed  "endocomple- 
ment"  was  destroyed  by  the  ether  just  as  were  the  serum  comple- 
ments. This;  however,  is  not  the  case.  When  the  blood  solution 
separates  after  shaking  with  ether  an  emulsified  stratum  is  formed 
between  ether  and  blood  solution.  On  testing  that  part  of  the  blood 
solution  which  contains  this  intermediary  stratum  the  entire  quantity 
of  the  activating  substance  is  recovered.  (See  Table  VI.) 

This  shows,  therefore,  that  the  activating  substance  had  not  been  de- 
stroyed, but  that  it  had  escaped  our  observation,  owing  to  the  peculiar 
behavior  of  the  emulsified  stratum.  It  is  well  known  that  such  emul- 
sions take  up  minute  solid  particles  most  readily,  and  it  was  natural 
therefore  to  assume  that  the  activator  contained  in  the  blood-cells 
is  connected  with  their  stromata.  In  laky  blood  solutions  the  stro- 
mata  are  present  in  a  swollen  state;  hence  we  sought  to  separate 
the  stromata  from  the  rest  of  the  hemoglobin  solution.  With  guinea- 


450 


COLLECTED   STUDIES   IN  IMMUNITY. 


pig  blood  solutions  this  is  very  simple,  for  on  strongly  centrifuging 
the  solution  used  for  complementing,  especially  if  some  salt  is  added, 
the  stromata  settle  out  very  well.  By  removing  the  supernatant 
haemoglobin  solution  and  perhaps  once  more  washing  the  sediment, 
the  stromata  are  readily  isolated.  This  suspension  of  blood  stromata, 
made  up  to  the  original  volume  with  salt  solution,  showed  itself 
just  as  capable  of  activating  cobra  venom  as  was  the  original  blood 
solution,  whereas  the  decanted  fluid  was  entirely  inert.  The  activating 
substance  of  the  blood  solution  is  present  therefore  not  in  solution  but 
as  a  constituent  of  the  stroma  of  the  blood-cells.  (See  Table  VII.) 

TABLE  VI. 


Ox  Blood  4-0.01  cc.  1%  Cobra  Venom  +  Blood  Solution. 

Amounts  of  the 

(6)  After  Shaking  with  Ether. 

Blood  Solution. 

(a)  Native 

II.   Upper  Half  of  the 

I    Lower  Half  of  the 

Blood  Solution  (to 

Blood  Solution. 

get  her  with  the  emulsi- 

cc. 

fied  stratum;. 

1.0 

complete 

0 

complete 

0.5 

1  ' 

0 

(  ( 

0.25 

4  « 

0 

t  « 

0.10 

" 

0 

i  < 

0.05 

0 

0 

it 

0.025 

0 

0 

faint  trace 

0.01 

0 

0 

0 

TABLE  VII. 


Amounts  of 


Ox  Blood +  0  01  cc.  1%  Cobra  Venom  + 


a.  b,  and  c. 
cc, 

(a)  Guinea-pig  Blood 
Solution. 

(6)  Suspension  of 
Blood-cell  Stromata 

(c)  Decanted  Por- 
tion. 

1.0 

complete 

complete 

0 

0.5 

0 

0.25 

<  ' 

(  i 

0 

0.15 

t( 

1  1 

0 

01 

little 

trace 

0 

0.05 

0 

0 

0 

This  threw  some  light  on  the  inactivation  of  the  blood  solution  at 
62°  C.,  a  fact  which  made  the  complement  character  of  the  acti- 
vating substance  seem  exceedingly  probable.  In  contrast  to  the 
native  blood  solution  we  find  that  the  suspension  of  stromata  remains 
unchanged  when  heated  to  62°  C. 


SUBSTANCES   WHICH   ACTIVATE  COBRA   VENOM. 


451 


The  activating  substance  itself  is  therefore  thermostable.  If, 
however,  the  decanted  haemoglobin  solution  is  again  added  to  the  stro- 
mata  and  this  mixture  heated  to  62°  C.  inactivation  will  again  ensue. 
(See  Table  VIII.) 

TABLE  VIII. 


Amounts  of 
a,  6.  and  c. 
cc. 

Ox  Blood  +001  cc    1%  Cobra  Venom  + 

(a)  Guinea-pig  Blood 
Stromata  Suspension. 

(6)  The  Suspension 
Heated  to  62°  C. 

(c)  The  Suspension  + 
Decanted  Fluid  (Hapmo- 
globin)  Heated  to  62°  C. 

1.0 

0.5 
0.25 
0.15 
0.1 
0.025 

complete 

« 

<  < 
1  1 

trace 
0 

complete 
<  « 

strong 
trace 
0 

0 
0 
0 
0 
0 
0 

From  this  it  appears  that  the  inactivation  of  the  native  blood 
solution  depends  on  this:  that  on  heating  to  62°  C.  the  active  substance 
combines  with  the  hemoglobin  in  such  fashion  that  it  is  no  longer 
able  to  combine  with  the  cobra  amboceptor.  Hence  in  view  of  the 
readiness  with  which  lecithin  combines  with  albuminous  substances, 
etc.,  we  believe  that  the  activating  property  of  dissolved  blood-cells 
which  we  previously  described  as  an  "endocomplement  action"  is 
really  due  to  the  presence  of  lecithin  or  lecithin-like  substances  in  the 
stroma.1 

We  have  convinced  ourselves  of  the  correctness  of  this  assump- 
tion also  by  the  fact  that  lecithin  is  bound  by  crystallized  horse, 
haemoglobin  by  heating  for  half  an  hour  to  62°  C.2  An  experiment 
of  this  kind  is  reproduced  in  Table  IX. 

A  solution  of  haemoglobin  heated  for  half  an  hour  to  62°  C.  is  also 
able  to  inhibit  the  activating  property  of  lecithin  when  digested  with 
this  for  half  an  hour  at  37°  C. 

The  lecithin  character  of  the  activating  substance  present  in  the 
red  blood-cells  is  confirmed  by  a  number  of  other  observations  which 
deal  with  the  analogous  character  of  cobra-venom  ha?molysis  on  the 

1  We  were  able  to  completely  extract  the  activating  substance  from  the 
stromata  suspensions  by  means  of  alcohol.     Besides  this,  in  activating  with 
stromata  in  the  presence  of  excess  of  cobra  venom,  one  observes  an  inhibition 
of  haemolysis  due  to  the  deflection  of  the  lecithin. 

2  We  are  much  indebted  to  Prof.  Hiifner  of  Tubingen  for  this  haemoglobin. 


452 


COLLECTED   STUDIES   IN    IMMUNITY 


addition  of  lecithin  and  of  blood  solution.     These  characteristics  are 
as  follows: 

1.  The  hsemolytic  activity  at  0°. 

2.  The  comparatively  rapid  course  of  haemolysis. 

3.  The  marked  inhibitory  action  of  cholesterin.     (See  Table  X.) 

TABLE   IX. 


Amounts  of 
the  Haemoglo 

Ox  Blood  +  0.01  cc.  1%  Cobra  Venom  + 
Haemoglobin-  Lecithin  Solution  .* 

bin-Lecithin 

Solution. 

cc. 

(a)  Native. 

(6)  Heated  for  One- 
half  Hour  to  62°  C. 

1.0 

complete 

0 

0.75 

'  ' 

0 

0.5 

i  ( 

0 

0.35 

little 

0 

0.25 

trace 

0 

0.15 

0 

0 

5  cc.  haemoglobin  X  5  cc   0.0125%  lecithin  solution. 

TABLE  X. 


Amounts  of 
the  Cholestenn 
Solution. 

cc. 

1  cc  5%  Ox  Blood  +  0.01  cc    1%  Cobra 
Venom  + 

(a)  0.25  cc.  Solution 
of  Guinea-pig 
Blood.t 

(6)  0.25cc.  001% 
Lecithin.  t 

0.025 
0.01 
0.005 
0.0025 

0 
trace 
moderate 
complete 

0 
0 
0 
complete 

t=complete  solvent  dose. 

It  will  be  remembered  that  in  these  three  points  guinea-pig  serum 
exhibited  exactly  the  opposite  behavior,  a  fact  which  led  us  to  ascribe 
its  activating  power  to  true  complements, 

We  have  therefore  come  to  the  conclusion  that  solution  by  means 
of  blood  solutions  is  only  a  property  of  the  lecithin  contained  in 
the  blood-cell  stroma,  and  is  not  due  to  true  complements.  We 
know  that  according  to  Ehrlich's1  conception  the  stroma ta  of  the 
red  blood-cells  are  to  be  looked  upon  as  living  protoplasm.  In  this 


1  Ehrlich,  Zur  Physiologic  und   Pathologic    der  Blutscheiben,  Charite   An- 
nalen,  Vol.  X,  1885. 


SUBSTANCES  WHICH   ACTIVATE  COBRA  VENOM.  453 

respect  the  demonstration  of  lecithin  in  the  stroma  would  appear  to 
be  of  special  interest,  for  just  this  substance  is  regarded  as  particu- 
larly important  for  the  functions  of  the  protoplasm.1 

A  further  problem,  to  be  sure,  is  whether  this  lecithin  exists  free 
in  the  red  blood-cells.  We  have  a  number  of  reasons  for  believing 
that  this  is  not  the  case.  It  was  first  shown  that  in  yolk  of  egg 
only  a  small  part  of  the  lecithin  can  be  shaken  out  with  ether,  whereas 
by  extracting  with  alcohol  the  entire  amount  can  be  obtained.2  The 
reason  for  this  is  that  the  greater  part  of  the  lecithin  is  conjugated 
with  the  vitellin  of  the  yolk.  This  combination  can  be  obtained 
as  a  globulin-like  body  which  is  soluble  in  salt  solution  and  precipitates 
on  dialyzing.3 

The  lecithin  is  obtained  free,  however,  only  after  extraction  with 
alcohol,  by  which  the  vitellin  also  changes  and  becomes  insoluble 
in  salt  solutions.  In  demonstrating  the  presence  of  lecithin  by 
means  of  cobra  venom  we  too  have  observed  that  the  serum  and  the 
red  blood-cells  yield  no  lecithin  to  ether,  or  if  they  do  it  is  only  in 
faint  traces.  On  the  other  hand,  the  active  power  of  the  alcoholic 
extracts  at  once  led  to  the  recognition  of  the  presence  of  lecithin. 

From  this  point  of  view  some  of  our  earlier  observations  can 
easily  be  explained.  We  stated  that  solutions  of  certain  species 
of  blood-cells  were  strongly  activating,  while  others  showed  this 
property  to  a  far  less  degree  or  not  at  all.  The  alcoholic  extracts 
of  all  species  of  blood,  however,  contain  nearly  the  same  amount  of 
lecithin  (demonstrated  by  the  activation  of  the  cobra  venom).  This 
apparent  contradiction  is  readily  explained  by  the  fact  that  in  the 
different  species  of  blood  the  lecithin  is  conjugated  with  different 
substances  of  the  stromata  and,  furthermore,  that  the  firmness  of 
this  combination  varies  extensively.  Thus  in  goat  blood  the  union 
is  so  firm  that  the  avidity  of  the  cobra  venom  does  not  suffice  to 
separate  the  two  components;  the  consequence  is  that  there  is  no 
activation  with  a  solution  of  goat  blood.  On  the  other  hand,  in 

1  It   has   long   been   known   that   lecithin   is  a  constant   constituent  of  the 
red  blood-cells;   for  many  species  of  blood-cells   this   content   has  even  been 
worked  out  quantitatively.     Nothing,  however,  was  thus  far  known  concern- 
ing the  localization  of  the  lecithin. 

2  See  Hoppe-Seyler's  Handbuch  der  physiologisch-  und  pathologisch-chem- 
ischen    Analyse,    Seventh   Edition,   edited    by    H.   Thierfelder,    Berlin,    1903, 
page  157. 

8  Ibid. ,  page  369. 


454  COLLECTED  STUDIES  IN  IMMUNITY. 

guinea-pig  blood,  for  example,  the  lecithin  is  so  loosely  combined 
that  this  blood  can  be  used  for  activation.  Hence,  in  speaking  of 
the  lecithin  action  of  animal  tissues  or  juices,  we  refer  only  to  the 
lecithin  which  is  free  (available)  in  the  sense  just  described;  part 
or  all  of  the  lecithin  present  may  escape  detection  by  means  of  the 
activation  of  cobra  venom. 

The  fact  that  relatively  slight  alterations  can  cause  the  combina- 
tion of  lecithin  to  be  either  looser  or  firmer  may  be  of  some  interest 
in  another  direction.  We  have  seen  that  the  lecithin  of  many  species 
of  sera  becomes  free  only  at  65°  C.,  while  the  haemoglobin,  on  the  other 
hand,  anchors  the  lecithin  at  62°  C.  It  is  possible  that  during  life 
slight  variations  in  the  physical  and  chemical  properties  of  the  tissues 
{variations  which  have  heretofore  been  undetected)  play  an  important 
role  in  the  sense  that  they  properly  regulate  the  exchange  and  trans- 
portation of  the  lecithin  so  important  for  the  vital  functions.  Dieu- 
donne's1  researches  show  that  the  albuminous  bodies  with  which 
the  lecithin  is  combined  (principally  in  the  form  of  lecithalbumin) 
are  demonstrably  modified,  even  at  temperatures  still  quite  distant 
from  their  coagulation  point.  This  author  showed  that  B.  coli;  for 
example,  when  inoculated  into  a  serum  lactose  solution  causes  a 
distinct  precipitation  even  at  45°  C.,  while  this  does  not  occur  at 
37°  C.  In  the  case  of  serum  albumin,  therefore,  the  temperature  at 
which  this  modification  takes  place  is  very  near  the  temperature 
which  occurs  in  the  living  organism  under  pathological  conditions, 
In  view  of  this  and  of  the  evident  dependence  of  the  physiological 
behavior  of  the  lecithin  on  the  integrity  of  the  albumin  molecule, 
one  is  tempted  to  see  a  causal  relationship  between  febrile  processes 
and  disturbances  in  lecithin  metabolism. 


HI.   The   Inhibitory  Action  of  Cholesterin. 

The  marked  inhibitory  action  which  many  sera  exert  on  haemolysis 
with  cobra  venom  and  lecithin  was  described  some  time  ago  (Kyes, 
1.  c.)  and  the  opinion  then  expressed  that  this  protective  action  of  the 
serum  was  probably  not  due  to  a  single  substance  but  was  the  resultant 
of  several  factors.  Evidently  we  are  here  dealing  with  certain  rela- 
tions which  exist  between  serum  constituents  and  the  lecithin,  making 

1  Dieudonne,  Uber  das  Verhalten  des  Bact  coli  zu  nativem  u.  denaturir- 
tem  Eiweiss,  Hyg.  Rundsch  1902,  No.  18. 


SUBSTANCES   WHICH  ACTIVATE  COBRA  VENOM.  455 

it  impossible  to  demonstrate  the  existence  of  the  latter  by  means 
of  cobra-venom  haemolysis.1 

Having  thus  learned  that  cholesterin  exerts  a  marked  inhibiting 
effect  on  the  action  of  lecithin  we  shall  probably  not  err  if  we 
assume  that  part  of  the  serum  protection  is  due  to  the  choles- 
terin present  in  the  sera.  One  thing  which  agrees  perfectly  with 
this  assumption  is  the  fact  that  often  this  protective  action  is  still 
present  after  heating  the  serum  to  100°  C. 

The  marked  inhibition  of  haemolysis  on  the  addition  of  choles- 
terin, an  inhibition  which  applies  also  to  the  haemolysis  produced 
by  lecithin  alone  when  in  large  quantities,  points  to  an  interesting 
antagonism  between  lecithin  and  cholesterin,  to  which  a  few  words 
may  be  devoted.2  In  this  case  the  cholesterin  probably  has  a  rela- 
tion to  lecithin  which  is  similar  to  that  of  saponin  in  Ransom's  well- 
known  experiments.3  In  both  cases  we  seem  to  be  dealing  with  the 
effect  of  a  kind  of  solvent  affinity  between  cholesterin  on  the  one 
hand  and  lecithin  and  saponin  on  the  other,  by  means  of  which 
affinity  the  presence  of  cholesterin  within  the  blood-cells  gives  rise 
to  toxic  action,  and  outside  of  the  erythrocytes  exerts  a  protective 
action.  It  is  possible  that  the  protection  observed  by  us  in  haemolytic 
test-tube  experiments  with  cholesterin  is  in  some  way  connected 
with  the  protective  action  of  cholesterin  against  snake  venom  in  the 
animal  body  described  by  Phisalix.4  Another  fact  may  be  men- 
tioned in  this  connection,  namely,  that  the  haemolysis  of  washed 

1  On  the  other  hand  the  specific  protection  exerted  by  Calmette's  snake- 
venom  immune  serum  is  not  an  antilecithin  effect,  but,  as  was  to  be  expected, 
one  depending  on  the  action  of  the  antibody  produced  by  immunization  (anti- 
amboceptors)  on  the  amboceptors  of  snake  venom.  When  varying  amounts 
of  lecithin  were  employed  the  protective  action  of  Calmette's  serum  remained 
constant,  always  neutralizing  the  same  amount  of  cobra  venom 

8  We  may  add  that,  like  Noguchi  (The  Antihsemolytic  Action  of  Blood  Sera, 
Milk,  and  Cholesterin  upon  Agaricin,  Saponin,  and  Tetanolysin,  etc.,  Univ.  of 
Penna.  Med.  Bulletin,  Vol.  XV,  No.  9,  1902),  we  observed  a  very  marked  choles- 
terin protection  against  the  action  of  tetanolysin.  (0.00025  cc.  of  our  stock 
solution,  which  certainly  contains  not  more  than  1%  cholesterin,  protects  against 
the  complete  solvent  dose  of  tetanolysin  (0.05  cc.).)  On  the  other  hand,  choles- 
terin is  absolutely  without  effect  on  the  hsemolyses  due  to  staphytolysin  and 
arachnolysin.  In  connection  with  this  we  might  mention  the  fact  so  inter- 
esting biologically,  that  even  so  indifferent  a  substance  as  neutral  olive-oil  dis- 
solves the  red  blood-cells.  This  haemolysis  is  likewise  inhibited  by  cholesterin. 

3  Ransom,  Saponm  und  sein  Gegengift.  Deut.  med.  Wochensch.  1901. 

4  Phisalix.  Compt.  rend,  de  la  Soc  de  Biologic,  1897. 


456 


COLLECTED  STUDIES  IN   IMMUNITY. 


guinea-pig  blood-cells,  in  themselves  susceptible  to  cobra  venom 
alone,  is  also  inhibited  by  cholesterin.  To  be  sure,  rather  large  quan- 
tities of  the  latter  are  required,  but  in  view  of  the  lecithin  character 
of  the  substances  which  functionate  as  endoactivators,  this  is  to  be 
expected.  (See  Table  XI.) 

TABLE  XI. 


Amounts  of  the 
Cholestedn  Solu- 
tion, cc. 

1  cc    5%  Guinea-pig 
Blood  X  0.0025  cc. 
1%  Cobra  Venom. 

1.0 
0.5 
0.25 
0.1 
0.05 
0.035 

0 
0 

little 
marked 
almost  complete 
complete 

On  the  other  hand,  as  already  remarked,  cholesterin  exerts  little 
or  no  protection  against  cobra-venom  haemolysis  when  serum  com- 
plement is  used  for  activation.  This  agrees  entirely  with  the  nega- 
tive findings  on  the  protective  action  of  cholesterin  recently  reported 
by  Flexner  and  Noguchi  in  an  interesting  paper  on  the  amboceptor, 
toxoids,  and  separate  constituents  of  snake  venom.1 

The  apparent  deviations  are  probably  to  be  explained  merely  by 
the  different  conditions  of  the  experiments,  for,  as  it  appears  to 
us,  these  authors  made  their  experiments  only  on  unwashed  blood- 
cells  or  by  the  addition  of  serum.  In  both  cases,  however,  one  is 
dealing  with  an  activation  with  complement,  against  which  we  also 
failed  to  detect  any  marked  protection  with  cholesterin. 

IV.   The    Quantitative    Relations    Existing  Between    Cobra  Venom 

and  Lecithin. 

So  far  as  the  mechanism  of  cobra-venom-lecithin  haemolysis  is 
concerned,  we  assume  that  the  lecithin  acts  after  the  manner  of 
complements,  being  anchored  by  certain  definite  groups  of  the  poison 
molecule.  This  has  previously  been  described  by  Kyes,  1.  c. 

Cobra  venom  and  lecithin  accordingly  combine  just  like  am- 
boceptor and  complement  in  serum  haemolysins,  and  it  was  there- 
fore to  be  expected  that  the  quantitative  relations  which  exist  be- 


1  Flexner  and  Noguchi,  The  Constitution  of  Snake  Venom  and  Snake  Sera, 
Univ  of  Penna.  Med.  Bulletin,  Vol.  XV,  No.  9,  1902. 


SUBSTANCES  WHICH   ACTIVATE   COBRA   VENOM 


457 


tween  amboceptor  and  complement  would  be  very  similar  in  this 
case.  In  our  studies  in  haemolysis  due  to  cobra- venom-lecithin  we 
have  therefore  been  able  to  observe  the  same  mutual  dependence 
between  amount  of  amboceptor  present  and  the  complement  re- 
quired which  the  researches  of  von  Dungern,1  Gruber  2  and  Morgen- 
roth  and  Sachs  3  showed  to  exist  in  their  experiments.  The  rela- 
tion between  these  amounts  is  such  that  when  large  amounts  of 
amboceptor  are  present,  smaller  doses  of  complement  suffice  for 
haemolysis. 

To  be  sure,  when  an  inordinately  large  amount  ot  cobra  venom 
is  added  the  amount  of  lecithin  required  for  complete  solution  is 
also  larger,  as  has  already  been  mentioned  by  Kyes.  This  is  evi- 
dently explained  by  assuming  that  when  the  amount  of  amboceptor 
is  excessive  the  distribution  of  the  lecithin  is  such  that  part  of  the 
amboceptor  loaded  with  lecithin  is  deflected  and  does  not  come 
into  action.  If,  however,  the  amount  of  cobra  venom  is  decreased^ 
results  will  be  obtained  which,  within  wide  limits,  agree  with  those 
observed  by  Morgenroth  and  Sachs  (1.  c.)  with  serum  haemolysins- 
The  more  cobra  venom  one  adds  the  less  lecithin  will  be  needed  to  effect 
complete  haemolysis,  and,  conversely,  in  adding  larger  amounts  of 
lecithin  the  minimal  complete  solvent  dose  of  the  cobra  venom  is 
constantly  decreased.  This  is  well  shown  by  Table  XII. 

TABLE  XII. 


1  cc.  5%  Ox  Blood. 


B. 

1  cc    5%  Ox  Blood. 


Amounts    of    the 
1%  Solution  of 
Cobra  Venom. 

The   Amount   of   Leci- 
thin Solution  (0.025%) 
Necessary  for  Com- 
plete Solution, 

Amounts  of  the  0  025% 
Lecithin  Solution. 

The  Amount  ot  Cobra 
Venom  (1%)  Neces 
sary  to  Effect  Com- 
plete Solution 

0.01 
0.001 

0.00025 
0.0001 
0.00001 

0.035 
0.05 
0.075 
0.1 

0.5 

0.3 
0.06 
'    0.03 

0.00001 
0.0001 
0.005 

From  these  experiments  we  see  that  the  quantitative  relations 
which  exist  between  cobra  venom  and  lecithin  furnish  an  additional 


1  von  Dungern,  page  36. 

2  Gruber,  Wiener  klin.  Wochensch.  1902,  No.  15. 

3  Morgenroth  and  Sachs,  pages  233  and  250. 


458 


COLLECTED  STUDIES  IN   IMMUNITY. 


argument  for  the  view  that  cobra  venom  and  lecithin  behave  like 
amboceptor  and  complement. 


V.  The  Susceptibility  of  the  Red  Blood-cells. 

These  observations  show  that  in  comparing  the  susceptibility  of 
the  various  species  of  blood  to  cobra  venom  the  limit  of  activity  of 
the  venom  must  be  determined  with  the  optimum  quantity  of  lecithin. 
The  values  thus  obtained  may  be  regarded,  so  to  speak,  as  the  " abso- 
lute susceptibility"  of  the  blood-cells.  In  Table  XIII  the  minimal 
complete  solvent  dose  is  determined  for  several  species  of  blood  on 
the  addition  of  an  abundant  quantity  of  lecithin  (0.2  cc.  of  a  0.025% 
lecithin  solution). 

TABLE  XIII. 


Species  of  Blood 
(1   cc.   of   a  5% 
Suspension). 

Amount  of  Lecithin, 
cc. 

Complete  Solvent 
Dose    of    Cobra 
Venom. 
Gram. 

Guinea-pig..  .  . 
Ox  

0.2  cc.  of  a  0.025%  sol. 

<  i 

0.00000005 
0.0000001 

Rabbit  

i  ( 

0.00000025 

Man   

•  (  i 

0  .  0000005 

Goat  

1  1 

0.000001 

If  we  compare  these  values  with  the  susceptibility  of  the  various 
blood-cells  with  cobra  venom  alone  (see  Table  XIV)  we  shall  see 
that  when  the  latter  is  used  the  amount  of  venom  necessary  for 
complete  haemolysis  is  many  times  greater  than  when  a  sufficient 
amount  of  lecithin  is  "added.  Thus  the  absolute  susceptibility  of 
guinea-pig  blood  against  cobra  venom  +  lecithin  is  500  times  greater 
than  that  obtained  without  the  addition  of  lecithin. 

This  shows  also  that  although  guinea-pig  blood  heads  the  list 
in  either  case  there  are  marked  deviations,  so  far  as  the  other  bloods 
are  concerned,  from  the  results  obtained  on  the  addition  of  lecithin. 
Ox  blood,  for  example,  which  is  not  at  all  susceptible  when  lecithin 
is  lacking,  is  more  susceptible  than  either  rabbit  or  human  blood 
when  lecithin  is  present.  Yet  the  two  latter  species  of  blood  are 
dissolved  even  without  the  addition  of  lecithin. 

We  thought  it  would  be  especially  interesting  to  study  the  sus- 
ceptibility of  human  blood-cells  to  cobra  venom  in  various  diseases. 
In  the  few  cases  thus  far  observed  (several  healthy  persons,  two  cases 


SUBSTANCES   WHICH   ACTIVATE  COBRA   VENOM. 


459 


of  diabetes,  one  of  pneumonia,  and  one  typhoid)  we  were  unable  to 
discover  any  essential  difference  in  susceptibility.1 

TABLE  XIV. 
SUSCEPTIBILITY  OF  VARIOUS  SPECIES  OF  BLOOD  TO  COBRA  VENOM  ALONE 


Species  of  Blood  (1  cc.5% 
Suspension). 

Amount  of  Cobra 
Venom  Required  tor  Com- 
plete Haemolysis. 

0.00001 

Doe 

0.000025 

Guinea-pig 

0  000025 

Man  

O.OOC05 

Rat  

0  00025 

Pig 

0.00025 

0.00025 

Goose  

0  .  0005 

Rabbit     

0.001 

Horse        

0.001 

Ox  sheep  goat  

not  susceptible 

As  a  result  of  our*  extensive  researches  we  must  continue  to 
uphold  the  view  that  blood  species  are  clearly  divisible  into  those 
directly  susceptible  to  cobra  venom  alone  and  those  not  susceptible 
under  those  conditions.  This  follows  also  from  the  above  table.  In 
this  respect  our  observations  are  at  variance  with  the  recent  state- 
ments of  such  excellent  workers  as  Flexner  and  Noguchi.  It  may 
be  well  therefore  once  more  to  point  out  a  few  possibilities  by  which 
this  difference  can  be  explained.  Flexner  and  Noguchi  observed 
that,  in  general,  after  copious  washing,  the  blood-cells  were  not  dis- 
solved by  cobra  venom,  or  at  least  were  only  partially  dissolved. 
In  spite  of  repeated  washing  of  the  blood  we  were  unable  to  discover 
any  decrease  in  susceptibility. 

If  Flexner  and  Noguchi  insist  on  such  a  thorough  washing  (6-10 
times)  it  appears  to  us  that  it  can  no  longer  be  a  question  of  removing 
the  serum  complements.  The  small  quantities  of  serum  which  are 
contained  in  the  0.05  cc.  blood  employed  in  each  tube  in  the  test- 
tube  experiment  (1  cc.  of  a  5%  suspension)  are  entirely  too  small, 
according  to  our  experience,  to  exert  a  demonstrable  complement 

1  It  is  possible  that  investigations  in  other  diseases  will  lead  to  positive 
results.  We  are  not  in  a  position  to  apply  our  observations  to  more  extensive 
clinical  material,  but  shall  be  glad  to  supply  cobra  venom  for  this  purpose 
to  any  one  applying  for  the  same. 


460 


COLLECTED   STUDIES  IN   IMMUNITY 


action  after  one  or  two  washings.  We  are  therefore  more  inclined 
to  assume  that  the  insusceptibility  observed  by  Flexner  and  Noguchi 
is  due  to  a  washing  out  of  the  activating  substances  present  in  the 
blood-cell.  One  of  us  has  already  reported  such  extraction  phenom- 
ena (Kyes,  1.  c.);  we  have,  however,  been  unable  to  repeat  the  ex- 
periments. It  is  possible,  as  has  already  been  stated,  that  the 
divergent  results  are  due  to  minute  differences  in  the  experiment, 
differences  which  for  the  present  at  least  cannot  be  analyzed.  It 
is  also  possible  that  a  certain  degree  of  racial  divergence  in  the  blood- 
cells  of  animals  of  the  same  species  used  by  Flexner  and  Noguchi 
and  by  us  gives  rise  to  what  at  present  is  an  inexplicable  difference. 
In  the  blood-cells  employed  by  us  the  activating  substances  could 
not  readily  be  washed  out.  This  is  shown  by  the  fact  that  the  acti- 
vating substances  are  so  firmly  bound  to  the  protoplasm  that  they 
are  not  separated  even  in  preparing  the  stromata. 

Attention  is  also  called  to  the  antagonism  which  is  so  often 
observed  between  blood-cells  and  their  own  serum.  This  has  already 
been  pointed  out  by  Kyes.  Thus  rabbit  blood-cells  are  dissolved 
by  cobra  venom,  and  this  action  is  intensified  by  the  addition  of 
rabbit  blood-cells  which  have  been  made  laky.  In  spite  of  this, 
however,  the  active  serum  of  the  same  rabbit  inhibits  cobra- venom 
haemolysis  (see  Table  XV).  In  this  case,  therefore,  adherent  traces 
of  serum  cannot  possibly  effect  autoactivation  of  the  rabbit  blood- 
cells. 

TABLE  XV. 


1  cc.  5%  Rabbit  Blood  + 

1%  Cobra  Venom, 
cc. 

Cobra  Venom  Alone. 

Cobra  Venom  +  0  05  cc. 
Rabbit  Serum. 

Cobra  Venom  +  0.05  cc. 
Rabbit-blood  Solu- 
tion (i). 

0.1 
0.05 

complete 
almost  0 

0 
0 

complete 
a 

0.025 

0 

0 

<  t 

0.01 

0 

0 

(( 

0.005 

0 

0 

n 

0.0025 

0 

0 

a 

There  is  another  point  of  considerable  interest  in  connection  with 
these  questions,  one  very  important  for  the  technique.  The  sus- 
ceptibility of  the  washed  blood-cells  can  readily  be  overlooked  in 


SUBSTANCES  WHICH   ACTIVATE  COBRA   VENOM. 


461 


many  cases  owing  to  the  occurrence  of  a  marked  inhibition  of  haemolysis 
due  to  the  presence  of  an  excessive  amount  of  cobra  venom. 

Kyes  (1.  c  )  has  already  discussed  in  detail  the  fact  that  in  hae- 
molysis with  cobra  venom  alone  a  phenomenon  can  occur  which  is 
analogous  to  the  deflection  of  complement  described  by  M.  Neisser 
and  Wechsberg.1  In  rabbit  blood  we  have  observed  extensive  indi- 
vidual differences  so  far  as  this  deflecting  phenomenon  is  concerned. 
We  have  often  found  animals  whose  blood-cells  remained  undissolved 
in  the  presence  of  even  a  very  slight  excess  of  cobra  venom,  so  that 
it  was  necessary  to  have  just  the  right  amount  of  venom  in  order 
to  effect  hamolysis.  Table  XVI  shows  several  examples  of  this. 

TABLE  XVI. 


Amounts  of  1% 

1  cc   5%  Rabbit  Blood. 

Cobra  Venom 

Rabbit 

Rabbit 

Rabbit 

Rabbit 

cc 

I. 

II. 

III. 

IV. 

1.0 

0 







0.5 

faint  trace 

— 

— 

— 

0.25 

little 

— 

— 

— 

0.1 

complete 

0 

trace 

complete 

0  075 

almost  0 

complete 

— 

«  « 

0  05 

0 

moderate 

marked 

<  < 

0.025 

0 

0 

complete 

t  ( 

0.01 

0 

0 

trace 

1  1 

0  005 

0 

0 

0 

strong 

0.0025 

0 

0 

0 

trace 

0.001 

0 

0 

0 

almost  0 

0  .  0005 

0 

0 

0 

0 

The  marked  deflection  which  is  observed  in  the  blood  of  rabbits  I, 
II,  III  is  evidently  caused  by  a  relatively  slight  amount  of  activating 
substances  present  in  and  at  the  disposal  of  the  red  blood-cells.  On 
the  other  hand  the  different  behavior  of  other  bloods,  as  in  rabbit  IV, 
shows  how  the  amount  of  free  lecithin  contained  in  the  blood-cells 
can  vary  from  case  to  case.  It  might  pay  to  examine  the  blood 
of  different  rabbits  for  this  purpose. 


See  page  120. 


462  COLLECTED  STUDIES  IN  IMMUNITY. 


VI.  A  Few  Chemical  Considerations. 

Finally,  we  should  like  briefly  to  discuss  some  of  our  experiences 
with  the  power  possessed  by  certain  other  substances  to  activate 
cobra  venom.  In  view  of  its  content  of  lecithin,  it  will  not  surprise 
us  to  know  that  bile  activates  cobra  venom.  It  may  be  interesting, 
however,  to  learn  that  goat  milk  acquires  activating  properties  only 
when  it  has  previously  been  heated  to  100°  C.  This  behavior  corre- 
sponds entirely  to  that  of  certain  species  of  sera  whose  lecithin  does 
not  become  available  until  after  they  have  been  heated  to  65-100°. 
Among  chemical  substances  we  have  found  a  number  of  fatty  acids 
and  their  soaps,  chloroform,  and  olive  oil  able  to  activate  to  a  cer- 
tain degree.  All  these  substances  by  themselves,  however,  dissolve 
the  blood-cells  to  a  greater  or  less  degree  l  and  the  increase  of  this 
action  is  so  slight  that  it  is  doubtful  whether  we  can  here  speak 
of  pure  activating  phenomena.2 

According  to  our  experiences  only  one  more  substance,  namely, 
the  lecithin-like  cephalin,  possesses  marked  activating  properties. 
(Cerebrin  does  not  possess  them.)  For  this  cephalin  we  are  indebted 
to  Waldemar  Koch  of  Chicago,  who  made  it  from  sheep's  brain. 
According  to  him,  it  is  a  dioxystearylmonomethyl  lecithin.3  The 
cephalin  (which  is  insoluble  in  alcohol)  and  the  lecithin  (which  is 
soluble  in  alcohol),  both  made  by  Koch  from  sheep's  brains,  further- 
more two  other  preparations  of  lecithin  (one  from  Riedel  in  Berlin, 
the  other  kindly  placed  at  our  disposal  by  Dr.  Bergell),  all  these 
manifested  a  hsemolytic  action  (if  at  all)  only  in  500-600  times  the 
amount  sufficient  to  activate  the  cobra  venom. 

A  preparation  of  lecithin  derived  from  leguminous  seeds,  for 
we  are  indebted  to  Prof.  Schulze  of  Zurich,  showed  less  dif- 
ference between  activating  power  and  hsemolytic  action,  but  even 


1  It  is  possible  that  the  coctostable  hsemolysins  (soluble  in  alcohol-ether) 
of  the  organ  extracts  belong  in  the  same  class  with  these  substances  (see 
Korschun  and  Morgenroth,  page  267). 

7  It  must  always  be  borne  in  mind  that  the  activating  property  of  these  sub- 
stances may  possibly  only  be  an  indirect  one,  the  presence  of  the  substance 
sufficing  to  make  available  the  lecithin  always  present  in  the  blood-cells  in 
combination. 

'  W.  Koch,  Zur  Kenntniss  des  Lecithins,  Cephalins  und  Cerebrins  aus  Nerven- 
substanz,  Zeitsch.  f  physiol.  Chemie,  Vol.  36,  Nos  2  and  3,  1903. 


SUBSTANCES  WHICH   ACTIVATE  COBRA   VENOM.  463 

in  this  the  ratio  was  still  1:200.  A  lecithin  obtained  from  E. 
Merck  behaved  similarly.  Nevertheless  all  of  these  preparations 
were  exactly  equal  in  their  activating  power.  It  is  hard  to  say  whether 
possibly  the  cholin  radical  or  the  fatty-acid  radical  represents  the 
active  toxophore  group  of  the  combination  formed  by  the  union  of 
the  lecithin  with  the  cobra  venom.  It  may  be  mentioned,  however, 
that  neutralized  cholin  exerts  no  hsemolytic  effect,  and  that  sinapin  l 
(the  sinapic  acid  ether  of  cholin),  despite  the  cholin  radical  which  it 
contains,  possesses  no  activating  power.  We  are  therefore  inclined 
to  believe  that  the  toxic  action  is  caused  by  the  fatty-acid  radical 
in  the  lecithin  molecule.  This  also  agrees  with  the  haemolytic  action 
observed  by  us  in  neutralized  stearylglycerophosphoric  acid  and  in 
the  above-mentioned  fats  and  fatty  acids.  We  shall  report  on  further 
researches  in  this  direction  at  a  subsequent  period. 

In  conclusion  we  may  be  permitted  to  discuss  briefly  a  few  inci- 
dental observations.  Among  these  is  the  fact  that  hydrochloric  acid 
not  only  causes  no  destruction  or  weakening  of  the  cobra  venom, 
but  even  exerts  a  marked  protective  action  on  the  same.  A  venom 
solution  which  completely  loses  its  activity  by  heating  to  100°  C. 
for  twenty  minutes  can  be  heated  for  half  an  hour  to  100°  C.  without 
losing  its  hsemolytic  property  if  it  contains  Vis^  HC1.  Not  until 
the  poison  containing  the  acid  is  heated  for  two  hours  to  100°  C. 
is  destruction  complete.  Possibly  the  protection  exerted  by  the  acid 
may  indicate  the  basic  character  of  those  binding  groups  of  the 
cobra-venom  molecule  which  are  here  concerned.  So  far  as  the 
influence  of  other  agents  on  the  cobra  venom  is  concerned  we  shall 
only  mention  that  all  procedures  which  prevent  the  action  of  the 
cobra  venom  in  the  animal  body2  (an  action  due  mainly  to  the 
neurotoxic  components  of  the  poison  3)  also  destroy  the  haemolytic 
action  of  the  venom.  Examples  of  this  are  powerful  oxidizing  sub- 
stances (potassium  permanganate,  chloride  of  lime,  chloride  of  gold, 
soda  lye,  etc.). 


1  For  this  we  are  indebted  to  Geheimrath  Schmidt  in  Marburg. 

2  See  especially  the  detailed  and  excellent  investigations  of  Calmette,  Annalea 
de  i'lnst.  Pasteur,  T.  VIII,  1894. 

*  See  Flexner  and  Noguchi,  1.  c. 


464  COLLECTED  STUDIES  IN  IMMUNITY. 


Resum^. 

1.  The  property  of  certain  sera  to  activate  cobra  venom,  a  prop- 
erty which  is  lost  by  heating  the  sera  to  56°  C.,  depends  on  the  presence 
of  complements  in  the  restricted  sense. 

2.  The   activating  property   of   blood  solutions   depends  on   the 
lecithin  contained  in  the  red  blood-cells;    this  also  gives  rise  to  the 
susceptibility   of   the  blood-cells   against   cobra   venom   alone.     The 
lecithin  which  comes  into  play  is  a  constituent  of  the  stromata. 

3.  The  fact  that  blood  solutions  are  inactivated  by  heating  to 
62°  C.  is  due  to  the  combination  at  this  temperature  of  the  lecithin 
with  the  haemoglobin;  suspensions  of  blood  stromata  are  not  inacti- 
vated at  this  temperature. 

4.  Cholesterin  inhibits  to  a  high  degree  haemolysis  by  means  of 
cobra    venom    alone,    and   of    cobra- venom-lecithin.      When    serum 
complements  are  used  for  activation,  cholesterin  exerts  little  or  no 
protective  action. 

5.  Cholesterin  does  not  inhibit  haemolysis  due  to  staphylolysin 
.and    arachnolysin,    but   very   markedly   inhibits   that   due   to   teta- 

nolysin  and  to  olive-oil. 

6.  The  quantitative  relations  between  cobra  venom  and  lecithin 
correspond  to  those  of  amboceptor  and  complement;   the  more  cobra 
venom  present  the  less  lecithin  will  be  required  for  haemolysis,  and 
vice  versa.     A  deflection  of  lecithin  does  not  occur  unless  very  large 
amounts  of  cobra  venom  are  used. 

7.  Most  species  of  blood  are  susceptible  even  to   cobra  venom 
alone.     The  "absolute  susceptibility"  determined  with  the  optimum 
addition  of  lecithin  may  be  many  times  that  obtained  without  the 
addition  of  lecithin. 

8.  Hydrochloric  acid  exerts  a  marked  protection  on  cobra  venom 
against    destruction    through    high    temperatures.      Potassium   per- 
manganate, chloride  of  lime,  chloride  of  gold,  soda  lye  destroy  cobra 
venom  (experiment  with  blood  +  lecithin). 

9.  Bile  activates  cobra  venom;  milk  (goat)  only  after  it  has  pre- 
viously been  heated  to  100°  C. 

10.  Fatty  acid,  soaps,  chloroform,  and  neutral  fats  have  a  haemo- 
lytic action.  The  haemolytic  action  is  somewhat  increased  on  the 
addition  of  cobra  venom. 

11.  Lecithin  and  cephalin,  on  the  other  hand,  exert  a  haemolytic 


SUBSTANCES  WHICH   ACTIVATE  COBRA   VENOM.  465 

action  on  the  ordinary  species  of  blood  only,  if  at  all,  when  200  or 
600  times  the  amount  is  used  which  suffices  for  activating  the  cobra 
venom. 

12.  In  the  poisonous  combination  formed  on  the  union  of  cobra 
venom  with  lecithin  the  fatty-acid  radical  may,  with  a  certain  degree 
of  probability,  be  regarded  as  the  active  group. 


XXXVI.   THE  ISOLATION  OF  SNAKE  VENOM 
LECITHIDS.1 

By  Dr.  PRESTON  KYES,  Instructor  in  Anatomy,  University  of  Chicago,  Fellow 
of  the  Rockefeller  Institute  for  Medical  Research. 

SPECIAL  interest  attaches  to  the  study  of  snake  venoms  be- 
cause of  the  analogy  which  exists  between  their  peculiar  character 
and  that  of  bacterial  toxins.  All  investigators  who  have  worked  with 
this  subject  have  been  struck  by  this  analogy,  and  Phisalix  2  has  dis- 
cussed it  in  a  special  monograph.  The  analogy  between  snake  venoms 
and  bacterial  toxins  consists,  above  all,  in  the  fact  that  neither  are 
crystallizable,  that  their  constitution  is  unknown,  that  both  are 
highly  virulent  specific  products  of  poison-forming  cells,  and  both 
possess  the  power  to  excite  the  production  of  antibodies  in  the 
organism.  This  last  fact  we  know  from  the  fundamental  researches 
of  Calmette.3 

A  further  analogy  between  snake  venoms  and  the  toxins  is  the 
fact  that  the  poisonous  properties  of  both  are  destroyed  by  heat, 
and  that  the  non-toxic  substance  thus  formed  is  able  to  excite  the 
production  of  antibodies  just  as  well  as  the  original  substance.  In 
other  words,  in  both  poisons  there  is  a  formation  of  toxoid.  Snake 
venom  has  accordingly  played  an  important  role  in  the  theoretical 
doctrine  of  immunity.  Martin  and  Cherry,4  for  example,  by  their 
well-known  filtration  experiment  were  able  to  prove  that  snake  venom 
and  specific  antitoxin  unite  to  form  a  new  non-poisonous  combination. 
This  experiment  is  based  on  the  principles  first  formulated  by  Ehrlich 

1  Reprint  from  the  Berliner  klin.  Wochensch.  1903,  Nos.  42  43. 

2  Phisalix,  Etude  compared  des  toxines  microbiennes  et  des  venins,  L'Annee 
biologique  I,  1895. 

3  Calmette,  Ann.  de  1'Institut  Pasteur,  No.  5,  1894. 

1  Martin  and  Cherry,  Proceedings  of  the  Royal  Society,  Vol.  LXIII,  1898. 

466 


THE  ISOLATION  OF  SNAKE  VENOM   LECITHIDS.  467 

in  his  studies  on  ricin  and  antiricin,  and  the  results  are  entirely  similar 
to  Ehrlich's.1 

Still  another  important  analogy  between  snake  venoms  and  bac- 
terial poisons  consists  in  their  plurality,  a  fact  which  has  been  demon- 
strated for  a  number  of  poisons.  In  the  ordinary  well-defined  chem- 
ical poisons  we  are  accustomed  to  regard  the  diverse  toxic  phenomena 
as  due  to  the  action  of  one  and  the  same  substance  on  different  organs. 
(In  poisoning  with  corrosive  sublimate,  for  example,  the  diverse 
toxic  phenomena  which  are  produced  in  the  various  organs.)  The 
toxins,  however,  have  to  a  large  extent  shown  a  different  behavior, 
the  action  on  different  organs  being  ascribed  to  different  kinds  of 
poisons,  which  frequently  possess  different  haptophore  groups.  The 
possibility  of  correctly  and  sufficiently  analyzing  these  poisons  de- 
pends in  a  large  measure  on  Ehrlich's  theory  of  the  combination  of 
these  poisons.  In  this  way  it  has  been  shown  that  tetanus  toxin 
consists  of  at  least  two  components,  tetanospasmin  and  tetanolysin,2 
to  which,  according  to  Tizzoni,  a  third  poison  must  be  added,  one 
which  gives  rise  to  the  cachexia. 

In  snake  venom  the  conditions  are  entirely  similar,  the  different 
effects  which  it  produces  in  the  animal  body  being  due  to  the  presence 
of  different  poisons  with  different  haptophore  groups.  The  late 
lamented  Myers  3  showed  that  the  haemoly tic  property  of  snake  venom 
is  to  be  separated  from  its  neuro toxic  property;  and  recently  Flexner 
and  Noguchi4  have  shown  that  the  oedematous  swellings  produced 
by  injections  of  snake  venom  are  due  to  the  presence  of  a  third  toxic 
component  acting  on  the  endothelium. 

For  some  years  I  have  closely  studied  cobra  venom,  and  especially 
that  constituent  of  the  same  which  causes  solution  of  the  red  blood- 
cells.  Part  of  these  researches  were  conducted  conjointly  with  Dr. 
H.  Sachs.5  I  was  able  to  confirm  the  interesting  observation  of 
Flexner  and  Noguchi  6  that  the  snake  venom,  as  such,  did  not  act 
on  certain  blood-cells,  but  that  hsemolysis  occurred  only  when  a  second 
substance  is  present  which  acts  after  the  manner  of  a  complement. 

1  Ehrlich,  Fortschritte  der  Medizin,  1897. 

2  Ehrlich  in  Madsen's  paper,  Zeitschrift  f   Hygiene,  Vol.  XXXII,  1899. 

3  Myers,  Journal  of  Pathology  and  Bacteriology,  1900,  VI,  405. 

4  Flexner  and  Noguchi,  Univ.  of  Penna.  Medical  Bulletin,  Vol.  XV,  No.  9, 
1902. 

6  Kyes,  see  page  291;   Kyes  and  Sachs,  see  page  443. 

8  Flexner  and  Noguchi,  Journal  of  Exp.  Medicine,  Vol.  VI,  No,  3.  1902. 


468  COLLECTED  STUDIES  IN  IMMUNITY. 

By  following  up  a  very  important  observation  made  by  Cal- 
mette,1  that  the  complementing  action  of  a  serum,  in  contrast  to 
what  is  seen  with  ordinary  complements,  is  still  preserved  after  heating 
to  62°  C.,  we  succeeded  in  discovering  what  this  complementing  agent 
was,  and  proved  that  lecithin  was  able  to  activate  the  cobra  venom 
amboceptor.  Especially  were  we  able  to  show  that  the  divergent 
behavior  ot  the  various  species  of  blood- cells  (some  of  them,  ox  blood, 
goat  blood,  sheep  blood,  are  not  dissolved  by  cobra  venom  alone, 
while  others,  such  as  guinea-pig  blood,  rabbit  blood,  human  blood, 
dog  blood,  are  dissolved  under  these  circumstances)  is  due  exclusively 
to  the  lecithin,  only  those  blood- cells  being  dissolved  in  which  the 
lecithin  is  so  loosely  bound  that  it  is  available  for  the  activation  of 
the  cobra -venom  amboceptor.2 

An  exact  study  of  these  activating  phenomena  by  means  of  lecithin 
seemed  to  us  to  be  of  the  highest  importance  for  one  of  the  fundamental 
problems  of  immunity,  namely,  the  mode  of  action  of  complements. 
Every  one  who  has  had  any  large  experience  with  the  activation  of 
ordinary  haemolytic  amboceptors  by  means  of  complements,  and 
who  compares  this  activation  with  that  of  cobra  venom  by  means  of 
lecithin,  will  be  surprised  at  the  complete  similarity  of  both  processes, 
and  will  not  doubt  that  essentially  the  same  mechanism  must  control 
both.  For  some  years  the  schools  of  Bordet  and  Ehrlich  have  had 
a  sharp  conflict  of  opinion  concerning  the  explanation  of  the  funda- 
mental facts  observed  by  Ehrlich  and  Morgenroth,  that  the  amboceptor 
is  anchored  by  the  red  blood- cells,  thus  making  the  blood-cells  sus- 
ceptible to  the  action  of  the  complements.  For  numerous-  reasons 
which  are  given  in  the  earlier  studies,  Ehrlich's  school  assumes  that 

1  Calmette,  Compt.  rend,  de  1'Acad.  des  Sciences,  T.  134,  No.  24,  1902. 

9  Some  time  ago  we  confirmed  the  observations  of  Flexner  and  Noguchi, 
that  blood-cells  unsusceptible  to  cobra  venom  alone  can  be  activated  by  certain 
fresh  sera,  and  that  this  activatibility  is  then  lost  on  heating  the  sera  to  56°  C. 
In  conformity  with  these  authors  we  assumed  that  the  cobra  venom  could 
also  be  activated  by  true  complements.  At  present,  however,  we  have  become 
rather  skeptical  as  to  the  correctness  of  this  explanation.  We  cannot  at  once 
dismiss  the  assumption  that  the  action  is  an  indirect  one,  the  action  of  the 
serum  causing  the  lecithin  combination  in  the  red  blood-cells  to  become  looser, 
so  that  then  this  substance  can  exert  its  activating  power  on  the  amboceptor. 
This  finds  further  support  in  several  observations  which  we  have  made  on 
the  favoring  influence  of  certain  indifferent  substances  (oils,  pure  fatty  acids) 
on  haemolysis  with  cobra  amboceptor.  In  these  cases  the  cause  of  the  solu- 
tion can  only  be  a  change  in  the  character  of  the  lecithin  combination. 


THE  ISOLATION   OF  SNAKE   VENOM  LECITHIDS.  469 

complement  and  amboceptor  unite  to  form  a  new  poisonous  combina- 
tion, and  this  is  the  view  which  I  also  take.  Bordet,  however,  even 
in  his  latest  study J  asumes  that  there  is  no  direct  affinity  between  com- 
plement (alexin)  and  amboceptor,  but  "que  la  sensibilatrice  modifie 
1 'element  de  maniere  a  lui  faire  acquerir  le  pouvoir  de  fixer  directement 
1'alexine  avec  beaucoup  d'energie." 

The  Frankfurt  Institute  has  furnished  a  number  of  important  argu- 
ments for  the  direct  union  of  amboceptor  of  complement.  Because 
of  the  lability  and  great  number  of  the  complements  as  well  as  the 
impossibility  to  isolate  the  active  product  chemically  it  was  out  of  the 
question  to  furnish  direct  chemical  proof  for  these  views.  Nor  is  there 
in  the  present  state  of  scientific  knowledge  any  hope  that  this  problem 
will  be  solved  in  the  near  future.  For  this  reason  we  rejoiced  in  the 
discovery  that  in  lecithin  we  had  found  a  substance  possessing 
complement-like  properties,  and  which  because  of  its  chemical 
behavior  would  serve  to  settle  this  dispute. 

In  other  words,  it  was  to  be  seen  whether  or  not  the  cobra  am- 
boceptor combined  directly  with  the  lecithin  to  form  a  new  haemolytic 
combination.  If  it  did  not  do  so,  it  would  help  sustain  Bordet's 
view  that  the  union  of  the  cobra  venom  amboceptor  serves  only  to 
give  the  lecithin  access  to  the  blood-cell.  From  the  following  studies 
it  will  be  seen  that  the  decision  which  we  had  been  led  to  expect 
as  the  result  of  our  biological  experiments  is  confirmed  by  chemical 
means. 

One  thing  especially  argued  for  the  correctness  of  our  conception, 
namely,  the  fact  that  it  is  possible  to  inhibit  the  cobra  venom 
hsemolysis  by  employing  very  large  amounts  of  cobra  amboceptor. 
In  that  case  susceptible  blood-cells  which  can  be  dissolved  by  a 
certain  definite  amount  of  cobra  amboceptor  are  no  longer  dissolved 
if  many  times  this  amount  is  employed.  This  corresponds  to  the 
phenomenon  which  we  observe  in  certain  bactericidal  sera,  and 
which  according  to  Neisser  and  Wechsberg  is  due  to  a  deflection  of 
complement  through  an  excess  of  free  amboceptor.  The  result  is 
comprehensible  only  on  the  assumption  of  a  direct  chemical  affinity 
between  amboceptor  and  complement.  For  this  reason  we  felt  that 
it  would  be  of  the  greatest  interest  to  gain  a  clearer  insight  through 

1  Mode  d' action  et  origine  des  substances  actives,  des  se*rums  preVentifs 
et  des  serums  antitoxiques.  Rapport  pre*sente"  par  J.  Bordet  au  Congr&s  de 
Hygiene  et  Demographic,  1903. 


470  COLLECTED  STUDIES  IN   IMMUNITY 

chemical  means  into  the  analogous  deflection  of  complement  observed 
with  cobra  amboceptor. 

1.  Preparation  of  Cobra  Lecithids. 

Owing  to  the  tenacious  character  and  the  slight  solubility  of  lecithin 
in  water  it  was,  of  course,  impossible  to  attempt  to  effect  the  desired 
combination  by  direct  mixture  of  the  aqueous  solution  of  cobra 
venom  with  lecithin.  On  the  contrary  it  was  necessary  to  adopt  a 
common  chemical  procedure,  " shaking  out,"  whereby,  through  the 
agency  of  an  appropriate  solvent,  the  lecithin  could  combine  with 
the  cobra  venom.  After  a  number  of  trials  we  found  the  best  solvent 
for  this  purpose  to  be  chloroform. 

In  our  experiments  we  employed  dried  cobra  venoms  which  had 
kindly  been  placed  at  our  disposal  by  Dr.  Lamb  and  Dr.  Greig  of 
Bombay,  and  Prof.  Calmette  of  Lille.  The  lecithin  used  was  the 
so-called  "Lecithol"  of  Riedel,  and  later  on  "Agfa-lecithin"  of  the 
Actien-Gesellschaft  fur  Anilin-Fabrication.  Both  of  these  proved  to 
be  excellent.  Special  emphasis  must  be  Jaid  on  a  sufficient  purity 
of  the  lecithin.  For  our  purposes  this  is  best  recognized  by  testing 
it  against  red  blood-cells.  0.5  cc.  of  a  1%  solution  of  the  lecithin 
should  not  dissolve  red  blood-cells.  If  the  contrary  is  the  case  the 
lecithin  should  be  purified  by  precipitating  it  once  or  twice  with 
aceton.1 

Forty  cc.  of  a  1%  solution  of  cobra  venom  in  a  85%  salt  solution 
are  mixed  with  20  cc.  of  a  20%  solution  of  lecithin  in  chloroform. 
The  mixture  is  placed  in  a  bottle  holding  about  100  cc.  and  thor- 
oughly shaken  for  two  hours  in  a  shaking  apparatus.  Thereupon 
the  mixture  is  centrifuged  for  three-quarters  of  an  hour  in  an  electric 
centrifuge  making  3600  revolutions  per  minute.  If  the  procedure 
has  been  successful  the  chloroform  layer  must  then  be  distinctly 
separated  from  the  watery  portion,  only  a  very  slight  compact, 
cloudy,  intermediate  layer  being  present.  If  the  lecithin  is  not 
sufficiently  pure  this  separation  will  not  take  place.  The  watery 
portion  is  separated  from  the  chloroform  layer  by  carefully  pipetting 
off  the  former.  The  chloroform  layer,  usually  measuring  about 

1  We  were  also  able  td  activate  cobra  amboceptor  with  a  brom-lecithin 
which  Dr.  Bergell  kindly  placed  at  our  disposal.  This  preparation  proved  less 
active  than  lecithin,  but  it  evidently  possesses  the  power  to  unite  with  cobra 
amboceptor  to  form  a  lecithid. 


THE  ISOLATIONS7   OF  SNAKE  VENOM  LEC1THIDS.  471 

19  cc.,  is  then  mixed  with  five  times  its  volume  of  chemically  pure 
ether  which  has  been  distilled  over  sodium.  A  precipitate  forms 
consisting  of  the  desired  cobra  venom  lecithid,  while  the  lecithin 
remains  dissolved  in  the  ether. 

Precipitate  and  fluid  are  separated  by  means  of  the  centrifuge, 
the  original  volume  of  ether  again  added,  shaken,  and  the  mixture 
once  more  centrifuged.  This  is  repeated  at  least  ten  to  twenty  times 
in  order  to  remove  any  adherent  lecithin.  The  substance  thus  ob- 
tained is  the  cobra  venom  lecithid. 

The  product  can  be  kept  for  a  long  time  under  ether,  apparently 
undergoing  little  or  no  change;  or  it  can  be  carefully  dried,  through 
which,  however,  it  suffers  some  change,  affecting  especially  its  solu- 
bility but  not  its  action.  The  yield  of  dry  substance  is  quite  large, 
1  grm.  of  dry  cobra  venom  yielding  about  5  grms.  dry  lecithid.1 

After  having  worked  out  the  best  method  for  obtaining  the  cobra 
lecithid  it  was  next  necessary  to  determine  by  biological  means 
whether  the  product  isolated  by  us  showed  itself  through  its  specific 
action  to  be  the  co bra-am boceptor-lecithin  combination  sought  for. 
That  this  was  actually  the  case  could  be  proven  in  two  ways, 
namely : 

1.  By  showing  that  the  extracted  watery  fluid  has  lost  its  hsemo- 
lytic  property,  and 

2.  By  showing  that  this  property  is  now  present  in  the  chloroform- 
lecithin  solutions  (see  Table  1). 

So  far  -as  the  behavior  of  the  aqueous  solution  is  concerned  it  can 
actually  be  shown  that  the  single  treatment  with  chloroform-lecithin 
removes  all  but  traces  of  the  haemolytic  power,  and  that  a  repetition 
of  the  procedure  suffices  to  remove  all  of  the  hsemolytic  agent  from 
the  watery  solution  Corresponding  to  this  we  find  that  the  haemo- 
lytic  power  of  the  watery  solution  has  been  transferred  completely 
to  the  chloroform-lecithin  portion,  a  fact  which  shows  that  the  leci- 
thin has  united  with  the  cobra  venom  (Table  I). 

1  If  one  has  but  very  little  of  the  primary  substance  at  one's  disposal  another 
method  of  preparing  the  lecithid  can  be  tried,  one  which  will  answer  at  least 
for  preliminary  examinations.  1  cc.  of  a  4%  -aqueous  solution  of  cobra  venom 
is  mixed  with  1  cc.  of  a  20%  solution  of  lecithin  in  methyl  alcohol,  the  mixture 
is  kept  in  an  incubator  for  several  hours  and  frequently  shaken;  then  10  cc. 
absolute  ethyl  alcohol  are  added  and  the  precipitated  albuminoids  separated 
by  filtration.  On  precipitating  the  clear  filtrate  with  ether,  one  obtains  the 
lecithid. 


472 


COLLECTED  STUDIES  IN   IMMUNITY. 


TABLE  I. 
1  cc.  5%  Ox  BLOOD +  0.2  cc.  0.1%  LECITHIN. 


cc. 

A. 
Native  0.001% 
Cobra  Venom 
(Control). 

B 

Cobra  Venom 
Shaken  Once 
with  01% 
Chloroform 
Lecithin 

B 
Cobra  Venom 
Shaken  Out 
Twice  with 
1.0% 
Lecithin- 
Chloroform. 

c 

Cobra  Lecithid 
from  Chloroform- 
Lecithin  by  Pre- 
cipitating with 
0.002% 
Ether1 

1.0 
0.75 

complete 

complete 

nil 
tt 

complete 

0.5 

<  « 

tt 

tt 

n 

0.35 

n 

tt 

tt 

tt 

0.25 

tt 

tt 

tt 

tt 

0.15 

i  i 

tt 

tt 

tt 

0.1 

almost  comp. 

»  < 

tt 

tt 

0.075 

marked 

1  1 



tt 

0.05 

little 

^  t 

— 

almost  complete 

0.035 
0.025 

trace 
almost  nil 

almost  comp. 
moderate 

— 

marked 
little 

0.01 

nil 

little 



trace 

0.0075 

tt 

trace 



almost  nil 

0.005 

tt 

almost  nil 



nil 

0.0035 

tt 

nil 

__ 

<  < 

0.0025 

tt 

tt 



•  « 

0.0015 

tic 

t  • 

— 

it 

Number   of  solvent 

doses    reckoned    on 

the  total  original  vol- 

ume of  40  cc 

266,000  to 

800 

0  0 

OAR  ftftO  to 

\J  •  \J 

£\J\J  y\J\J\J       L\J 

267,000 

267,000 

Percentage  of  haemoly- 

sins  in  each  solution  . 

100% 

0.003% 

0.0% 

100% 

1  In  comparison  to  the  original  aqueous  solution  of  venom. 

The  assumption  that  the  cobra  amboceptor  is  extracted  by  chloro- 
form alone  is  refuted  by  control  tests. 

The  neurotoxic  action  of  the  native  poison  is  entirely  absent  in 
the  cobra  lecithid.  Relatively  large  quantities  of  the  lecithid  in 
aqueous  solution  can  be  injected  subcutaneously  into  animals  with- 
out producing  constitutional  symptoms.  For  example,  an  amount 
of  lecithid  which  suffices  to  destroy  200  cc.  mouse  blood  can  be 
injected  into  mice  weighing  15  grms.  without  causing  any  further 
symptoms  than  infiltration  at  the  site  of  injection.  In  like  manner 
rabbits  can  be  injected  subcutaneously  with  10  cc.  of  a  1%  solution 
of  the  lecithid  without  causing  any  constitutional  symptoms.  In  this 
case,  however,  the  local  reaction  is  extensive,  the  infiltrated  area 
often  including  a  considerable  portion  of  the  abdominal  surface. 


THE   ISOLATION   OF  SNAKE  VENOM  LECITHIDS. 


473 


According  to  this,  therefore,  the  second  constituent  of  cobra 
venom  does  not  pass  into  the  chloroform-lecithin.  In  this  respect, 
however,  we  have  been  able  to  demonstrate  that  the  watery  portion 
which  has  practically  been  freed  from  the  haemoyltic  amboceptor  still 
possesses  its  toxic  properties  in  animal  experiments.  (See  Table  II.) 
The  essential  difference  between  the  haemo  toxin  and  the  neuro toxin , 
first  pointed  out  by  Myers,  is  thus  confirmed  by  direct  chemical 
means. 


TABLE  II. 

COMPARATIVE  TEST  OF  THE  NEDROTOXIC  ACTION  OF  A  SOLUTION  OF  COBRA 

VENOM   (a)   BEFORE  AND   (6)  AFTER  SHAKING  THE  COBRA   AMBOCEPTOR 

WITH  CHLOROFORM-LECITHIN. 


0.01%  Venom. 

(a)  Native   Venom, 

(6)  Extracted  Venom. 

0.5 
0.35 
0.25 
0.15 
0.12 
01 

t  after  2  hours 
f     "     2J  hours 
f     -     1|     " 
t     "     2i     " 
t     "     30-40  hours 
living 

t  after  1  hour 
t     "     IJ  hours 
t     "       Jhour 
t     "     8  hours 
t     "     30-40  hours 
living 

II.  The  Properties  of  Cobra  Lecithids. 

In  the  description  of  cobra  lecithid  we  shall  do  best  to  keep  to 
the  product  obtained  by  the  method  above  described,  the  last  traces 
of  lecithin  having  been  removed  from  the  ethereal  precipitate  by 
repeated  washing  with  ether,  and  the  main  portion  of  the  ether  in 
turn  removed  by  pressing  the  precipitate  between  two  folds  of  filter- 
paper. 

This  primary  product  is  insoluble  in  aceton  and  ether,  but  soluble 
in  chloroform,  in  alcohol  (cold),  and  in  toluol  (on  heating).  The 
solutions  in  chloroform  and  in  alcohol  are  precipitated  by  the  addi- 
tion of  ether  and  aceton.  When  still  moist  with  ether  it  dissolves 
very  readily  in  water,  a  point  of  some  importance.  Even  if  the 
ether  which  the  product  contains  is  rapidly  evaporated  by  means 
of  a  current  of  air  and  the  product  then  dissolved  in  water  an  abso- 
lutely clear,  light-yellow  solution  will  be  obtained. 

These  facts  show  that  the  primary  product  is  absolutely  different 
from  the  two  substances  from  which  it  was  derived,  cobra  ambo- 
ceptor and  lecithin.  It  differs  from  lecithin  particularly  in  its  insolu- 


474  COLLECTED  STUDIES  IN   IMMUNITY 

bility  in  ether  and  its  ready  solubility  in  water;  from  cobra  venom 
amboceptor  in  its  solubility  in  the  above-mentioned  organic  solvents, 
alcohol,  chloroform,  toluol.  Cobra  venom  does  not  give  up  even  a 
trace  of  cobra  amboceptor  to  these  solvents. 

It  has  been  found  that  the  watery  solution  of  the  primary  cobra 
lecithid  obtained  from  cobra  venom  and  lecithin,  as  described  above, 
undergoes  spontaneous  modification  which  leads  to  the  formation 
of  an  insoluble  substance.  If  the  watery  solution  is  allowed  to  stand 
at  room  temperature  it  gradually  becomes  cloudy,  and  in  the  course 
of  a  few  hours  a  whitish  precipitate  is  formed.  On  removing  this  pre- 
cipitate, either  by  filtration  or  by  centrifuge,  a  precipitate  will  again 
form  in  the  clear  fluid.  The  sediment  is  microcrystalline,  white,  trans- 
parent, and  very  refractile. 

It  can  easily  be  shown  that  this  sediment  is  nothing  but  a  modified 
form  of  the  lecithid,  for  after  thoroughly  washing  the  precipitate 
which  has  been  separated  by  the  centrifuge,  it  will  be  found  that 
this  still  exerts  its  full  hamolytic  action.  In  accordance  with  this, 
the  original  solution  of  the  primary  product  shows  a  proportionate 
loss  of  power.  In  one  experiment  which  we  followed  rather  closely 
we  found  that  in  course  of  time  about  two-thirds  of  the  lecithid  had 
separated  out  in  solid  form,  while  one-third  was  still  left  in  solution. 
The  secondary  lecithid  produced  in  this  way  is,  as  already  stated, 
almost  insoluble  in  cold  water;  on  the  other  hand,  it  is  readily  soluble 
in  warm  water,  although  it  again  separates  on  cooling.  This  be- 
havior constitutes  the  chief  difference  between  the  primary  and  the 
secondary  lecithid;  the  behavior  of  the  two  substances  toward  the 
above-mentioned  organic  solvent  is  identical 

Owing  to  its  character  the  secondary  lecithid  is  particularly  adapted 
for  chemical  investigations,  and  one  of  the  foremost  authorities  has 
already  commenced  work  on  this  substance.  Some  important  results 
which  have  already  been  obtained  will  be  mentioned  later  on.  For 
the  present  we  shall  merely  mention  that  the  product  gives  no  biuret 
reaction  even  when  in  concentrated  solutions.  We  are  reserving  for 
future  study  the  chemical  study  of  the  above  lecithids,  as  well  as  the 
investigation  of  the  neurotoxin  obtained  in  purified  form  by  means 
of  the  method  above  described. 

The  formation  of  the  secondary  lecithid  also  occurs  if  the  ethereal 
precipitate  is  dried  at  incubator  temperature.  It  is  then  easy  to  see 
that  such  a  product  has  more  or  less  completely  lost  its  solubility  in 
water,  especially  if  it  has  remained  in  the  thermostat  for  several  days. 


THE  ISOLATION  OF  SNAKE   VENOM  LECITHIDS.  475 

In  its  properties  the  insoluble  portion  corresponds  entirely  with  the 
secondary  lecithid  precipitate  from  aqueous  solutions.  To  obtain 
the  secondary  lecithid  as  a  pure  product,  however,  the  method  first 
mentioned  seems  preferable,  namely,  that  which  starts  with  the  aque- 
ous solution  of  the  primary  substance;  the  result  seems  to  be  a  lighter- 
colored  product. 

It  is  natural  that  this  lecithid  when  finished  differs  in  its  action 
in  many  ways  from  the  cobra  amboceptor.  It  can  readily  be  under- 
stood that  the  cobra  lecithid  acts  on  the  blood-cells  of  all  the  species 
thus  far  examined,  no  matter  whether  these  cells  possess  available 
lecithin  or  not.  One  fact  of  considerable  interest  has  been  discov- 
ered, namely,  that  the  absolute  quantity  of  lecithid  necessary-  for 
haemolysis  is  the  same  for  the  blood-cells  of  different  species.  We 
found  that  an  amount  of  lecithid  which  corresponded  to  about  0.003 
mg.  dry  cobra  venom  was  able  to  dissolve  1  cc.  of  a  5%  suspension 
of  blood-cells  of  different  species  (guinea-pig,  rabbit,  man,  ox).  This 
quantity,  we  may  add,  corresponds  to  the  amount  of  cobra  venom 
which  causes  solution  of  the  blood -cells  in  the  ordinary  test  when  a 
large  excess  of  lecithin  is  present. 

An  observation  which  is  also  of  considerable  interest  is  a  com- 
parison of  the  time  necessary  for  the  action  of  the  cobra  lecithid  and 
the  amboceptor,  with  and  without  the  addition  of  lecithin.  In  our 
previous  papers  we  pointed  out  that  when  cobra  venom  is  allowed 
to  act  on  susceptible  blood-cells,  solution  occurs  after  a  considerable 
period  of  incubation,  so  that  in  case  a  minimal  quantity  is  employed 
12  to  18  hours  (two  hours  at  37°,  then  at  8°)  elapse  before  complete 
solution  is  effected.  Even  if  a  suitable  excess  of  cobra  venom  and 
the  most  susceptible  species  of  blood  are  employed,  at  least  10  to 
30  minutes  will  usually  elapse  before  solution  is  completed.  Similar 
differences  are  observed  if,  as  previously  described,  we  allow  cobra 
venom  and  lecithin  to  act  on  unsusceptible  blood-cells.  In 
that  case,  again,  with  minimal  quantities  of  lecithin  and  ambo- 
ceptor 6  to  18  hours  are  necessary  to  effect  solution;  this  time 
is  decreased  if  large  excesses  are  employed,  but  solution  is  never 
instantaneous. 

In  contrast  to  this  a  marked  decrease  in  the  period  of  incubation 
is  observed  if  the  finished  lecithid  is  used,  solution  being  instantaneous 
on  the  employment  of  cencentrated  solutions.  The  shortening  in 
the  time  necessary  for  solution  becomes  particularly  marked  when 
small  doses  of  the  lecithid  are  used,  solution  commencing  at  once 


476 


COLLECTED  STUDIES   IN    IMMUNITY. 


and  being  completed  within  15  to  20  minutes.     In  other  words,  the 
increase  in  the  rapidity  of  the  process  is  about  twenty-fold. 

This  behavior  is  significant,  for  it  shows  that  in  this  case  the  period 
of  incubation  is  due  not  to  a  slow  action  of  the  anchored  toxophore 
group  (lecithin),  but  exclusively  to  the  slow  development  of  the  real 
toxic  agent,  the  lecithid.  The  difference  in  the  time  of  action  in  the 
case  of  small  and  large  doses  is  in  accordance  with  the  well-known 
law  that  the  reaction  (in  this  case  the  union  of  cobra  arnboceptor 
and  lecithin)  proceeds  more  rapidly  in  concentrated  solutions  than 
in  weak  ones. 

TABLE  III. 


1  cc.  5%  Ox  Blood. 

Amounts  of 
Cholesterin 

A. 

Solution.' 

Native  Cobra  Venom, 
about  1*  Solvent 
Doses  with  the  Addi- 
tion of  Lecithin. 

. 
Primary  Cobra  Lecithid, 
about  H  Solvent 
Doses. 

. 

Secondary  Cobra 
Lecithid,  about 
1*  Solvent  Doses. 

0.1 

0 

0 

0 

0.075 

0 

0 

0 

0.05 

0 

0 

0 

0.035 

0 

0 

0 

0.025 

0 

0 

0 

0.015 

0 

almost  0 

almost  0 

0.01 

0 

trace 

trace 

0.0075 

0 

little 

little 

0.005 

almost  0 

moderate 

moderate 

0.0035 

trace 

marked 

marked 

0.0025 
0.0015 

little 
moderate 

almost  complete 
complete 

almost  complete 
complete 

0.001 

marked 

0.00075 

<  < 

0.0005 

11 

0.00035 

<  < 

0.00025 

almost  complete 

0.00015 

complete 

*The  solution  of  cholesterin  was  made  by  diluting  1  cc.  of  a  saturated  solution  of  Cholesterin 
in  hot  methyl  alcohol,  with  9  cc.  85%  salt  solution 

A  third  difference  between  cobra  amboceptor  and  the  finished 
lecithid  is  seen  in  the  behavior  toward  high  temperature.  The  aqueous 
solution  both  of  the  primary  and  the  secondary  cobra  lecithid  is 
far  more  stable  than  solutions  of  the  amboceptor  alone.  The  former 
can  be  heated  to  100°  C.  for  six  hours  without  any  particular  loss  in 
power,  while  the  amboceptor  of  cobra  venom  loses  its  action  if  heated 
to  100°  C.  for  only  thirty  minutes.  Obviously  this  is  to  be  explained 


THE   ISOLATION   OF  SNAKE  VEN7OM   LEC1TH1DS.  477 

by  assuming  that  the  combination  has  become  firmer  by  the  entrance 
into  it  ot  the  lecithin  molecule. 

There  is  a  fourth  point  of  difference,  the  behavior  toward  the 
snake-venom  serum  discovered  by  Calmette.  The  finished  lecithid 
is  affected  far  less  by  this  serum  than  is  the  cobra  amboceptor.  We 
shall  discuss  this  in  a  later  article. 

In  contrast  to  these  differences  the  behavior  of  cobra  lecithid  and 
cobra  amboceptor  +  lecithin  toward  cholesterin  is  similar.  We  have 
already  mentioned  that  cholesterin  possesses  the  power  to  inhibit 
the  haemolysis  by  means  of  cobra  venom.  The  same  is  true  in 
haemolysis  by  means  of  the  finished  lecithid,  although  quantitatively 
to  a  less  degree.  (See  Table  111  opposite.) 

IV.  The  Lecithids  of  Several  Other  Poisons. 

Naturally  it  was  of  considerable  interest  to  see  whether  this 
peculiar  formation  of  lecithid  (thus  far  without  parallel  in  chemistry) 
was  confined  to  cobra  venom,  or  extended  also  to  other  poisons.  The 
following  poisons,  which  we  owe  to  the  courtesy  of  Dr.  Lamb,  Prof. 
Calmette,  Dr.  Kinyoun,  Dr.  Dowson,  and  Prof.  Kitasato,  have  there- 
fore been  studied  by  us  for  this  purpose: 

1.  Bothrops  lanceolatus; 

2.  Daboia  Russettii; 

3.  Naja  haye; 

4.  Kerait; 

5.  Bungarus  fasciatus; 

6.  Trimeresurus  anamalensis  (Hill  viper) ; 

7.  Trimeresurus  Riukiuanus  (Japan); 

8.  Crotalus  adamantus. 

In  a  subsequent  article  we  shall  discuss  the  behavior  of  these 
poisons  toward  different  species  of  blood-cells.  For  the  present,  how- 
ever, we  may  say  that  all  of  these  poisons,  on  the  addition  of  suffi- 
cient lecithin,  dissolve  the  blood-cells  examined  by  us,  namely,  those 
of  man,  guinea-pig,  rabbit,  ox.  With  the  exception  of  two  poisons 
(Bothrops  lanceolatus  and  Trimeresurus  anamalensis)  the  absolute 
quantity  of  poison  necessary  to  effect  solution,  an  excess  of  lecithin 
being  present,  is  about  the  same  for  all  species  of  blood  examined; 
0.003  grm.  are  sufficient  to  dissolve  1  cc.  of  a  5%  suspension.  The 


478  COLLECTED  STUDIES  IN   IMMUNITY. 

Bothrops  poison  is  ten  times  weaker,  and  that  of  Trimeresurus  anama- 
lensis  twenty-five  times.  This  observation  made  the  formation  of 
a  lecithid  seem  probable.  As  a  matter  of  fact  it  was  easy,  by  means 
of  the  method  above  described,  to  prepare  a  solid  lecithid  which 
contained  the  entire  haemolytic  power  of  the  poisons.1  Hence  we 
believe  that  in  general  all  haemolytic  snake  venoms  are  of  the  am- 
boceptor  type  and  possess  a  lecithinophile  group,  the  occupation  of 
which  by  lecithin  gives  rise  to  the  haemolytic  action.  In  fact  it 
seems  as  though  in  the  last  analysis  the  factor  which  determines  the 
type  of  the  haemolytic  action  of  snake  venom  was  principally  this 
lecithinophile  group. 

A  fact  which  goes  to  support  this  view  is  the  observation  that 
several  of  the  poisons  examined  by  us  probably  differ  in  their  hapto 
phore  group,  which  unites  with  the  receptor  of  the  blood-cells.  Thus 
Lamb2  has  shown  that  the  Daboia  amboceptor,  unlike  the  cobra 
amboceptor,  is  not  inhibited  in  its  action  by  Calmette's  serum.  The 
same  is  true  for  Bothrops,  Crotalus,  and  Trimeresurus  Riukiuanus, 
whereas  the  poisons  of  Bungarus  and  Naja  haye  are  similar  to  the 
cobra  venom  so  far  as  their  behavior  toward  the  serum  is  concerned. 

It  is  quite  possible,  therefore,  that  the  differences  in  the  various 
types  of  poison  are  only  differences  in  the  haptophore  group,  while 
the  characteristic  lecithinophile  group  is  identical  in  all  cases. 

It  was  important  to  see  whether  in  animals  other  than  snakes 
poisons  are  present  which  are  capable  of  forming  lecithids.  We 
therefore  next  studied  the  poison  of  the  scorpion,  choosing  this 
because  Calmette  3  had  already  shown  that  the  acute  fatal  action 
of  scorpion  poison  could  be  inhibited  by  the  snake-venom  serum, 
a  fact  indicating  a  certain  analogy  between  the  toxic  components 
of  scorpion  poison  and  snake  venom.4  We  were  able  to  determine 
that  the  scorpion  poison  by  itself  exerts  only  a  slight  haemolytic 
action  on  guinea-pig  blood-cells,  leaving  other  species  of  blood-cells 
unaffected.  On  the  addition  of  lecithin,  however,  it  exerts  con- 

1  In  conformity  with  its  weaker  action  Bothrops  poison  yields  only  a  tenth 
the  lecithid  obtained  from  the  other  poisons,  and  the  poison  of  Trimeresurus 
anamalensis  only  one  twenty-fifth. 

2  Lamb,  Scientific  Memoirs,  Medical  and  Sanitary  Depts.,  Govt.  of    India, 
1903,  No.  3. 

3  Calmette,  Ann.  de  ITnstit.  Pasteur,  1895,  No.  4. 

4  For  this  scorpion  poison  we  are  much  indebted  to  Prof.  Treub,  Director  of 
the  Botanical  Garden  in  Buitenzorg. 


THE  ISOLATION   OF  SNAKE  VENOM  LECITHIDS. 


479 


siderable  solvent  action  on  all  the  different  species  of  blood  examined 
by  us.  Its  action  is  about  one  twentieth  as  strong  as  that  of  cobra 
venom.  (See  Table  IV.) 

TABLE  IV. 
ACTION  OF  SCORPION  POISON  WITH  AND  WITHOUT  THE  ADDITION  OF  LECITHIN. 


Amounts  of  the  0.2% 
Solution  of 

1  cc-  5%  < 

3x  Blood. 

Scorpion  Poison 
cc. 

+  0.2  cc.  0.1% 
Lecithin. 

Control  without 
Lecithin. 

1.0 

complete 

0 

0.75 

0 

0.5 

0 

0.35 

0 

0  25 

0 

0.15 

0 

0.1 

0 

0.075 

0 

0.05 

0 

0.035 

0 

0  025 

. 

0 

0.015 

almost  complete 

0 

0  01 

moderate 

0 

0.0075 

little 

0 

0.005 

trace 

0 

0.0035 

1  1 

0 

0  0025 

faint  trace 

0 

0.0015 

0 

0 

Corresponding  to  this  behavior  we  succeeded  in  actually  pro- 
ducing a  typical  lecithid  from  scorpion  poison  by  following  the  usual 
procedure.1 

All  this  leads  us  to  the  view  that  the  essential  character  of  the 
haemolytic  cobra  venom  is  due  not  to  the  haptophore  group,  but 
finally  to  the  lecithin  anchored  by  the  blood-cells  by  means  of  a 
lecithinophile  amboceptor.  Now  we  know  that  lecithin  is  present 
in  every  red  blood-cell,  and  this  seems  apparently  to  contradict  the 
fact  determined  by  us  experimentally  that  the  lecithin  is  the  cause 
of  haemolysis.  This  contradiction,  however,  is  merely  apparent, 
for  we  need  only  assume  that  by  the  aid  of  the  cobra  venom 

1  It  is  probable  that  the  poison  of  a  fish,  Trachinus  draco  (see  Briot,  Journ. 
de  Physiol.  et  de  Pathol.  ge"n.  1903,  No.  2),  is  also  capable  of  forming  a  lecithid; 
at  least  a  statement  of  Briot  speaks  in  favor  of  this,  namely,  that  the  haBmolytic 
agent  in  the  Trachinus  poison  can  be  activated  by  a  serum  which  has  been 
heated  to  more  than  60°  C. 


480  COLLECTED  STUDIES  IN  IMMUNITY. 

the  lecithin  is  brought  into  proximity  with  cell  constituents  other 
than  those  normally  in  its  proximity.  In  other  words,  we  are  dealing 
with  the  deleterious  action  of  a  vitally  important  substance  which 
has  been  forced  into  the  wrong  place.  This  conclusion  is  made  plain 
if  we  bear  in  mind  the  fact  that  in  the  blood-cells  primarily  suscep- 
tible to  cobra  amboceptor,  the  ha?molytic  action  depends  not  on  the 
addition  of  new  lecithin,  but  on  a  transposition  of  the  lecithin  pre- 
formed in  the  cell,  due  to  the  anchoring  of  the  cobra  amboceptor. 


XXXVII.    THE   CONSTITUENTS    OF   DIPHTHERIA 

TOXIN.1 

By  PAUL  EHRLICH. 

THE  Festschrift,  published  at  the  opening  of  the  Serum  Institute 
in  Copenhagen,  contains  a  study  by  Arrhenius  and  Madsen 2  which 
deals  mainly  with  the  neutralization  phenomena  of  toxin  and  anti- 
toxin. We  must  all  rejoice  that  Madsen  has  succeeded  in  interest- 
ing so  excellent  a  physical  chemist  in  this  question,  especially  as  I 
had  tried  unsuccessfully  for  years  to  secure  the  interest  of  physical 
chemists  in  Germany.  In  the  present  state  of  scientific  knowledge 
we  shall  for  the  present  have  to  give  up  our  attempts  to  isolate  the 
toxins  in  pure  form.  For  the  same  reason  also  in  the  analysis  of  the 
relations  between  toxin  and  antitoxin  we  cannot  conform  to  the 
ordinary  methods  of  the  chemist  working  with  the  balance.  On  the 
other  hand,  the  study  of  toxin  and  antitoxin  is  of  too  great  practical 
importance  for  us  to  wait  idly  for  years  or  decades  until  chemistry 
is  so  far  advanced.  We  must,  therefore,  content  ourselves  with  the 
slight  means  at  our  disposal,  applying  these,  however,  in  all  direc- 
tions in  order  to  gain  as  great  an  insight  into  this  complicated 
subject  as  the  present  state  of  our  knowledge  permits.  I  had  ap- 
plied myself  to  this  problem  for  years  and  come  to  the  conclusion 
that  the  only  way  to  approach  it  was  by  an  exact  quantitative  study 
of  the  neutralization  phenomena.  Particularly  in  partial  neutraliza- 
tion I  believed  I  had  found  a  method  by  which  we  could  gain  an 
insight  into  the  most  intricate  constitution  of  the  toxins.  To  my 
regret  high  authorities  pronounced  this  method  as  incorrect  and  of 
no  avail.  I  am  all  the  more  pleased,  therefore,  to  see  that  so  high 

1  Reprint  from  the  Berl.  klin.  Wochenschr.  1903,  Nos.  35-37. 

2  S.  Arrhenius  and  Th.  Madsen,  Physical  Chemistry  applied  to  Toxins  and 
Antitoxins,  Festkrift  ved  indvielsen  af  Statens  Serum  Institute,  Kopenhagen, 
1902.     (This  is  to  be  had  in  English  text,  Kopenhagen,  1902.) 

481 


482  COLLECTED  STUDIES   IN   IMMUNITY 

an  authority  as  Arrhenius  recognizes  my  method  as  correct  in  prin- 
ciple and  proceeds  along  the  same  lines. 

The  study  of  Arrhenius  and  Madsen  deals  principally  with  tetano- 
lysin, the  hsemolytic  poison  discovered  by  me  in  tetanus  toxin.  Tetano- 
lysin  and  tetanospasmin  differ  from  each  other  in  their  haptophore 
groups,  as  a  result  of  which  each  possesses  a  particular  antibody 
in  the  tetanus  serum  of  the  market.  Madsen  studied  this  tetano- 
lysin  in  my  Institute,  and  found  that  when  it  is  gradually  neutralized 
with  increasing  amounts  of  antitoxin,  the  same  definite  amounts  of 
antitoxin  first  added  neutralize  far  more  poison  than  subsequent 
additions.  Because  of  this  and  also  because  of  other  reasons  (atten- 
uation, phenomena  during  neutralization)  Madsen  concluded  that 
several  poisons  of  different  affinities  were  present. 

On  taking  up  these  studies  in  tetanolysin  Arrhenius  and  Madsen 
obtained  practically  the  same  results,  and  these  authors  succeeded 
in  constructing  a  formula  for  the  action  of  antitetanolysin  on  tetano- 
lysin which  conforms  to  the  law  of  Guldberg-Waage.  Based  on 
this  they  next  attempted  to  determine  similar  relations  in  the  case 
of  very  simple  blood  poisons.  This  had  already  been  done  by  Danysz, 
but  the  method  was  open  to  criticism.  Arrhenius  and  Madsen  chose 
a  weak  base  and  an  acid  (ammonia  and  boric  acid)  as  hsemolysin 
and  antihaemolysin.  It  was  found  that  in  these  the  neutralization 
phenomenon  is  very  similar  to  that  of  tetanolysin  and  antilysin, 
from  which  they  concluded  that  in  the  neutralization  of  toxins  and 
antitoxins  we  are  dealing  with  reactions  between  simple  substances 
of  weak  affinities. 

In  this  connection  they  express  themselves  as  follows:  "The 
last-mentioned  curve  gives  a  fairly  accurate  picture  of  the  neutraliza- 
tion of  ammonia  with  boric  acid.  In  the  investigation  of  ammonia 
as  hsemolysin  a  spectrum  analogous  to  that  of  toxin  or  tetanolysin 
(Fig.  3)  could  have  been  constructed ;  the  following  conclusion  could 
also  perhaps  have  been  drawn:  One  part  of  boric  acid  (antitoxin) 
added  to  ammonia  neutralizes  50%  of  this  base;  if  two  parts  are 
added  it  neutralizes  66.7%;  if  three  parts,  75%;  and  four  parts, 
80%.  From  this  it  follows  that  since  the  respective  amounts  50, 
16.7,  8.3,  and  5%  are  each  time  neutralized  by  the  same  amount 
of  boric  acid,  the  amount  first  neutralized  is  three  times  as  toxic  as 
the  amount  next  neutralized,  this  again  twice  as  toxic  as  the  next 
after  it,  which  in  its  turn  is  one  and  one-half  times  as  toxic  as  the 
following,  etc.  In  other  words,  ammonia  is  not  a  simple  substance, 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN  483 

but  consists  of  several  constituents  of  different  toxicity  (and  these 
toxicities  bear  a  simple  reciprocal  relation  to  each  other).  Of  these 
constituents  the  toxin  possessing  the  highest  chemical  affinity  is 
neutralized  first. 

A  similar  conclusion  has  actuall}1  been  drawn  in  the  case  of  toxin ; 
the  toxin  first  neutralized  (the  strongest)  has  been  called  prototoxin, 
the  next  deuterotoxin,  the  next  tritotoxin,  etc.  The  final  very 
weak  toxins  are  called  toxones." 

The  findings  of  Arrhenius  and  Madsen  are  thus  seen  to  be  directly 
opposed  to  my  statement  that  diphtheria  poison  is  composed  of 
several  constituents.  In  view  of  the  exceeding  importance  of  the 
subject  I  cannot  avoid  entering  the  discussion  and  state  the  reasons 
which  cause  me  to  maintain  my  views  absolutely  and  without  any 
modification.1 

The  new  views  of  the  authors  in  question  will  doubtless  lead  many 
to  wonder  how  I  could  err  in  so  simple  a  matter  and  employ  compli- 
cated theories  when  the  simplest  conceptions  of  chemistry  would  have 
sufficed.  It  must  seem  strange  that  I,  who  have  followed  this  sub- 
ject for  years  and  have  busied  myself  especially  with  chemical  studies, 
should  have  failed  to  discover  this  very  ready  explanation.  As  a 
matter  of  fact,  however,  I  too  began  with  the  conception,  now  held 
by  Arrhenius  and  Madsen,  that  in  the  union  of  toxin  and  antitoxin 
we  were  dealing  with  a  phenomenon  of  incomplete  neutralization.  A 
more  thorough  analysis  of  diphtheria  poison  (my  publications  refer 
only  to  this  poison)  compelled  me,  however,  to  adopt  more  complex 
explanations. 

At  the  very  outset  of  my  investigations  I  discovered  that  tetano- 
lysin  and  its  antitoxin  possess  weak  affinities,  and  I  devised  the  tech- 

1  Gruber,  whose  experiments  especially  devised  to  refute  my  theory  1  was 
able  to  show  were  incorrect,  has  employed  the  opportunity  to  side  with 
Arrhenius  and  Madsen,  and  to  announce  that  their  observations  "will  give 
this  entire  spook  of  side-chain  theory  its  quietus,"  No  one  who  knows  any- 
thing about  this  subject  needs  be  told  that  the  question  as  to  whether  diph- 
theria poison  is  made  up  of  one  or  more  substances  has  nothing  to  do  with  the 
side-chain  theory.  When  1  formulated  this  theory  I  too  believed  the  diph- 
theria poison  to  be  a  simple  substance,  and  when  subsequently  1  felt  compelled 
to  differentiate  several  components  in  the  poison  1  always  emphasized  that 
the  separate  components  differed  only  in  their  toxophore  group  and  were 
similar  so  far  as  the  haptophore  groups  were  concerned,  the  groups  which  give 
rise  to  antitoxin  formation  (see  my  reply  to  Gruber  in  Munch,  med.  Wochenschr 
1903,  Nos.  33  and  34). 


484  COLLECTED  STUDIES  IN  IMMUNITY. 

nique  of  my  experiments  accordingly.  At  that  time  I  stated  in  connec- 
tion with  this  tetanolysin  that  the  union  of  toxin  and  antitoxin  pro- 
ceeds more  slowly  in  dilute  solutions  than  in  concentrated,  and  that 
the  process  is  hastened  by  heat.  How  feeble  the  combining  affinities 
of  tetanus  toxin  and  antitoxin  are  can  be  seen  from  the  following 
experiment  devised  over  eight  years  ago:  If  a  given,  not  very  con- 
centrated, mixture  of  serum  and  toxin  is  allowed  to  stand  for  two 
hours  it  will  be  found  that  the  action  of  the  serum  is  forty  times  as 
great  as  when  the  mixture  is  employed  immediately.  Whether  the 
optimum  of  neutralization  is  thus  reached  is  difficult  to  say.  The 
determination  of  the  exact  limits  fails  because  of  the  fact  that  the 
poison  rapidly  decomposes  in  watery  solutions,  especially  if  these 
be  dilute.  One  constantly  faces  either  of  two  difficulties :  insufficient 
union  on  the  one  hand  and  decomposition  of  the  poison  on  the  other.1 
With  diphtheria  poison,  on  the  other  hand,  the  affinity  of  the 
toxin  for  the  antitoxin  is  much  greater.  As  is  well  known,  these 
substances  unite  so  rapidly  that  even  the  time  for  combination 
prescribed  in  the  test — fifteen  minutes — is  still  unnecessarily  long. 
Hence,  even  if  I  admit  that  the  union  of  tetanolysin  and  antilysin 
is  comparable  to  the  neutralization  of  a  weak  base  by  a  weak  acid, 
I  shall  in  the  following  pages  show  that  the  affinity  of  diphtheria 
toxin  and  antitoxin  is  very  great,  comparable  perhaps  to  that  between 
a  strong  acid  and  base.  In  accordance  with  this  also  I  am  convinced 
that  the  neutralization  of  diphtheria  toxin  by  antitoxin  proceeds  in 
the  form  of  a  straight  line  and  not  in  that  of  a  curve.  This,  then, 
constitutes  my  first  objection  to  the  general  deductions  drawn  by 
Arrhenius  and  Madsen  from  their  particular  findings.  Just  as  it  is 
impossible  to  apply  the  results  of  the  neutralization  of  boric  acid  and 
ammonia  to  every  combination  of  acid  and  base,  so  it  is  impossible 
to  apply  the  experiences  with  tetanolysin  to  the  doctrine  of  toxins 
in  general.2 


1  When,  then,  years  ago,  in  spite  of  these  unfavorable  conditions,  I  proposed 
the  study  of  tetanolysin  to  Thorvald  Madsen,  this  was  but  a  makeshift  neces- 
sitated by  the  lack,  at  that  time,  of  suitable  hsemolysins.     At  present  a  number 
of  such  substances  are  available,  such  as  arachnolysin  and  snake  venom.     These 
are  very  stable  and  far  better  suited  for  exact  determination  since  the  factor 
of  decomposition  is  absent. 

2  1  should  like  to  mention  that  recently  Dr.  Kyes  has  discovered  that  in 
snake  venom  also  the  neutralization  with  antitoxin  proceeds  with  high  affinities 
and  in  a  straight  line. 


THB  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  485 

If,  therefore,  the  affinity  between  diphtheria  toxin  and  antitoxin 
is  so  great,  we  shall  have  to  ascribe  the  irregular  course  of  the  neutraliza- 
tion process  to  other  factors  than  those  assumed  by  Arrhenius  and 
Madsen. 


Diphtheria  Toxins. 

In  order  to  understand  what  follows  it  will  be  necessary  to  speak 
of  some  of  the  main  principles  of  toxin-antitoxin  analysis.  As  is 
well  kno>Tn  diphtheria  toxin  is  the  bouillon  fluid  in  which  the  diph- 
theria bacilli  have  grown,  and  to  which  they  have  given  up  their 
toxic  secretory  products.  In  order  to  determine  the  toxicity  we 
make  use  of  guinea-pigs.  The  lethal  dose  (L.  D.)  is  that  amount 
of  poison  which  will  surely  kill  a  guinea-pig  weighing  250  grammes 
on  the  fourth  day.  In  order  to  determine  the  relations  between  toxin 
and  antitoxin  it  is  best  to  start  from  the  serum  because  this  can  be 
preserved  constant  by  means  of  the  methods  devised  by  me  (vacuum, 
drying).  This  dry  serum  also  serves  as  the  standard  for  the  officia 
titration.  The  immune  unit  (I.  E.  =  Immunitats  Einheit)  is,  of  course, 
an  arbitrary  quantity  which  originated  by  terming  that  amount  of 
antitoxin  a  unit  which  just  neutralized  100  L.  D.  of  a  poison  that 
happened  to  be  available  at  the  time,  so  that  the  mixture  when  injected 
did  not  produce  even  the  slightest  trace  of  illness  (either  general  or 
local  reaction). 

If  one  mixes  one  immune  unit  of  serum  with  graduated  amounts 
of  poison,  two  limits  may  be  obtained.  One  of  these  is  termed 
limit  zero  (L0),  and  corresponds  to  the  quantity  of  poison  which  is 
completely  neutralized  by  1  I.  E.  The  other  is  limit  death  (Lt)  and 
corresponds  to  that  quantity  of  poison  which  on  the  addition  of  1  I.  E* 
is  so  far  neutralized  that  only  just  one  L.  D.  remains.  Of  these  two 
limits  the  Lt  is  very  easily  and  accurately  determined  so  that  it 
serves  as  a  measure  in  testing  the  potency  of  the  diphtheria  serum. 
This  limit  signifies  nothing  more  than  that  of  x  L.  D.  present,  1  I.  E. 
neutralized  x-l  L.  D.,  so  that  just  1  L.  D.  remains  free  and  leads 
to  the  death  of  the  guinea-pig  in  four  days. 

A  priori  one  might  have  expected  that  the  number  of  lethal  doses 
which  are  neutralized  by  1  I.  E.  is  always  the  same  in  poisons  from 
different  sources.  The  only  difference  which  one  would  have  ex- 
pected would  be  that  in  different  poison  solutions,  the  volume  in 


486  COLLECTED  STUDIES  IN  IMMUNITY. 

which  a  given  number  of  L.  D.  were  contained  would  vary  from  case 
to  case,  depending  on  the  varying  quantity  of  poison  produced  by 
the  bacilli. 

Closer  investigations,  however,  showed  that  in  reality  the  con- 
ditions are  entirely  different,  the  number  of  L.  D.  contained  in  Lt 
varying  enormously  in  different  toxic  bouillons.  In  poisons  which 
have  been  analyzed  the  figures  have  fluctuated  between  15  and  160. 
Since  it  had  been  shown,  especially  by  myelf,  that  the  neutralization 
of  toxin-antitoxin  rests  on  a  chemical  basis,  this  result  could  only 
be  explained  by  assuming  that  the  diphtheria  bouillon,  in  addition 
to  the  toxins,  contained  other  non-toxic  substances  which  were  able 
to  combine  with  antitoxin  just  like  the  diphtheria  toxin.  I  deemed 
it  to  be  of  the  highest  importance  to  clear  up  this  mystery  experi- 
mentally, and  therefore  subjected  a  number  of  different  poisons  (some 
freshly  derived,  others  precipitated  with  ammonium  sulphate,  and 
still  others  which  had  been  kept  for  a  long  time)  to  comparative 
analyses.  In  the  course  of  these  it  was  found  that  the  non-toxic 
substances,  which  still  possess  combining  properties,  increase  as  the 
toxic  bouillon  ages,  and  I  therefore  studied  these  changes  in  the 
poisons  genetically  at  various  stages. 

I  emphasize  this  part  of  my  method  because  the  casual  remark 
by  Arrhenius  and  Madsen  1  that  my  results  were  derived  mainly  from 
a  study  of  decomposed  poisons  might  readily  be  misconstrued  and 
give  one  the  impression  that  in  my  investigations  I  had  not  been  espe- 
cially careful.  I  may  at  once  add,  however,  that  my  most  valuable 
results  were  obtained  by  studying  the  course  of  this  decomposition, 
but  this,  of  course,  corresponds  entirely  with  the  methods  of  chem- 
istry. It  is  impossible  to  gain  an  insight  into  the  constitution  of 
highly  complex  combinations  by  means  of  an  analysis  which  leads 
only  to  the  compact  formula.  This  can  only  be  gained  by  the 
careful  decomposition  of  the  substance  to  be  studied.  Whatever 
knowledge  we  possess  regarding  the  constitution  of  sugars,  uric  acid 
derivatives,  alkaloids,  etc.,  is  due  mainly  to  the  decompositions  intel- 
ligently carried  out,  and  a  careful  study  of  their  products.  Of  course, 
the  decomposition  must  not  give  rise  to  secondary  reactions  which 
could  obscure  the  results ;  this  might  be  the  case  if  strong  acids  or  a 
high  temperature  were  employed.  The  decomposition  must  be 
quantitative  and  of  moderate  intensity.  The  following  observa- 
tions will  show  that  this  is  especially  the  case  in  the  spontaneous 

'I.e. 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIX.  487 

attenuation  of  the  toxins,  which  occurs  at  room  temperature  and 
without  any  further  chemical  manipulation.1 

It  has  been  found  that  the  bouillon  on  standing  can  preserve  its  neutral- 
izing property  intact,  and  often  actually  does  so,  while  the  toxicity  is 
considerably  decreased.  Observations  of  this  kind  have  been  made 
by  myself  and  Madsen  for  diphtheria  poison,  by  Jacoby  for  ricin, 
by  Myers  for  snake  venom,  and  recently  by  Arrhenius  and  Madsen 
for  tetanus  poison.  This  phenomenon,  which  in  many  cases  is  quanti- 
tative, is  most  readily  explained  by  assuming  that  the  poison  molecule 
contains  two  functionating  groups.  One,  the  "haptophore  group," 
combines  with  the  antitoxin  and  in  the  animal  body  effects  the  com- 
bination with  the  tissues;  this  group  is  quite  stable.  The  other, 
the  "toxophore  group/'  effects  the  true  poisonous  action;  it  is  com- 
paratively readily  destroyed.  In  my  opinion  the  transformation  of 
toxin  into  toxoids  by  the  destruction  of  the  toxophore  group  is  the 
key  to  a  correct  understanding  of  my  conception  of  antitoxic  im- 
munity and  the  subject  of  toxins.2 

If  we  see,  for  example,  that  in  spite  of  decreased  toxicity  the 
constants  of  neutralization  Lf  and  L0  remain  entirely  unchanged, 
it  follows,  in  my  opinion,  that  two  important  deductions  can  be  made. 
The  first  is  one  which  I  have  always  drawn,  namely,  that  in  normal 
toxoid  formation  not  brought  about  by  chemical  additions,  the  num- 
ber of  haptophore  groups  suffers  no  loss.  This  behavior,  however, 
also  seems  to  indicate  that  in  toxoid  formation  the  affinity  of  the  hapto- 
phore groups  for  the  antitoxin  is  in  no  way  changed.  I  may  be  per- 
mitted to  elucidate  this  by  means  of  a  chemical  example.  Tetra- 
methylammoniumhydroxid  is  a  very  strong  base  (like  KOH)  which 
through  suitable  procedures  (heating,  etc.)  is  transformed  into  the 

1  Obviously  these  poisons  can  also  be  attenuated  through  chemic  or  thermic 
influences,  but  the  decomposition  in  that  case  takes  place  rapidly  and  with 
destruction.  In  my  investigations,  therefore,  I  have  never  made  use  of  these 
methods,  but  have  kept  to  the  moderate  changes  which  occur  spontaneously 
in  the  toxic  bouillon  on  standing. 

7  At  the  outset  of  the  modern  study  of  immunity,  von  Behring,  Aronson, 
and  others  had  observed  that  an  active  immunity  could  be  brought  about 
particularly  through  attenuated,  modified  poisons.  At  that  time,  however, 
it  was  very  difficult  to  appreciate  these  relations,  and  so  in  the  year  1894  we 
find  a  high  authority,  as  a  result  of  his  investigations,  denying  the  existence 
of  modified  poisons,  although  he  had  previously  assumed  their  existence.  The 
results,  which  had  been  obtained  with  immunization,  he  ascribed,  not  to  the 
presence  of  modified  poisons,  but  exclusively  to  a  dilution  of  the  poison. 


488  COLLECTED  STUDIES  IN  IMMUNITY. 

far  less  basic  trimethylamin,  methyl  alcohol  being  split  off  in  the 
process.  Let  us  take  a  certain  definite  quantity  of  tetramethylam- 
monium  hydroxid,  say  20  molecules,  and  determine  the  quantity 
of  boric  acid  which  will  just  suffice  for  complete  neutralization,  as 
shown  by  a  suitable  indicator.  On  changing  the  ammonium  base 
into  the  tertiary  amin  (a  change  which  we  shall  assume  to  be  com- 
plete) we  shall  find  that  a  larger  quantity  of  boric  acid  is  necessary 
for  neutralizing  the  tertiary  amin.  In  other  words,  there  has  been 
a  change  in  the  position  of  the  neutral  point,  although  the  number 
of  basic  radicals  remains  the  same.  This  necessarily  follows  from  the 
decrease  in  affinity  brought  about  by  the  transformation. 

The  reverse  will  take  place  if  a  weak  base  is  transformed  into  a 
stronger  one.  A  change  in  the  position  of  the  neutral  point  will  occur 
even  if  the  transformation  is  only  a  partial  one,  i.e.,  does  not  affect 
the  entire  number  of  molecules.  If,  however,  in  spite  of  an  extensive 
formation  of  toxoid,  we  find  the  test  limits  unchanged,  we  can  only 
conclude  that  any  considerable  change  in  affinity  has  not  occurred. 
We  shall  subsequently  learn  of  another  fact,  which  affords  conclusive 
evidence  of  the  correctness  of  these  views. 

Our  next  problem  will  be  to  study  the  influence  of  the  toxoids 
on  the  neutralizing  process.  To  begin,  it  should  be  remarked  that 
the  bacterial  poisons  with  which  we  are  dealing  are  not,  as  a  rule, 
pure  poisons.  By  this,  of  course,  I  do  not  mean  to  deny  that  pure 
poisons  can  occur.  If  the  toxophore  group  possesses  considerable 
resistance  so  that  it  is  not  affected  by  the  processes  used  in  its  pro- 
duction (keeping  in  the  incubator  for  weeks,  etc.),  it  will  be  possible 
to  obtain  poisons  which  contain  only  toxins  and  no  toxoids.  Such  a 
result,  however,  can  probably  only  be  counted  on  in  a  small  number 
of  isolated  cases,  and  is  not  obtained  as  a  rule.  So  far  as  diphtheria 
poison  is  concerned,  of  which  I  have  made  a  special  study,  I  have 
never  yet,  among  a  large  number  of  specimens  examined,  found  a 
single  one  free  from  toxoids.  In  estimating  the  degree  of  purity 
one  proceeds  by  finding  in  various  poisons  how  many  fatal  doses 
(L.  D.)  are  neutralized  by  one  immune  unit  (I.  E.).  The  maximum 
value  in  the  poisons  at  my  disposal  was  130,  but  Madsen  has  described 
a  poison  in  which  the  Lf  dose  contained  160  L.  D.  But  even  this 
poison,  as  I  shall  show  later,1  merely  approached  the  character  of  a  pure 
poison. 

1  It  is  especially  important  that  even  diphtheria  poisons  which  have  been 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN  489 

Naturally  the  poisons  whose  toxophore  groups  are  very  labile 
will  be  the  least  pure.  This  is  especially  true  in  tetanus  poison, 
which  is  far  more  readily  destroyed  than  diphtheria  poison.  In  the 
former,  several  hours'  standing  of  an  aqueous  solution  suffices  to  give 
rise  to  toxoid  formation.  It  is  all  the  more  probable,  therefore, 
that  the  toxin  produced  in  the  usual  manner  by  keeping  the  culture 
in  the  incubator  for  eight  days  contains  a  considerable  admixture  of 
toxoids.  In  the  precipitation  with  ammonium  sulphate  these  tox- 
oids,  of  course,  are  present  in  the  resulting  solid  product. 

A  dry  poison  of  this  kind,  such  as  I  placed  at  Madsen's  disposal 
for  his  experiments,  can,  of  course,  keep  for  a  long  time  unchanged 
provided  it  is  carefully  preserved;  the  primary  content  of  toxoid, 
however,  also  remains  unchanged. 

For  this  reason  I  believe  that  the  assumption  of  Arrhenius  and 
Madsen,  that  the  tetanus  poison  used  by  them  was  a  pure  poison, 
since  it  did  not  change,  is  entirely  unwarranted.  It  is  even  possible 
that  this  particular  specimen  contained  far  more  toxoids  than  the 
old  toxin  solutions  which  I  had  employed. 

In  pure  chemistry  in  carrying  out  exact  mathematical  determina- 
tions it  is  a  general  principle  that  the  substance  be  either  absolutely 
pure  or  at  least  that  its  degree  of  purity  be  exactly  determined  by 
analysis.  In  determining  the  molecular  weight  of  an  element,  a  great 
deal  of  preliminary  work  (recrystallization,  etc.)  is  required  in  order 
to  obtain  the  original  material  as  pure  as  possible.  If  this  cannot 
be  done,  as,  for  example,  in  the  case  of  hydrogen  peroxide,  or  ozone, 
a  quantitative  study  requires  at  least  that  the  exact  percentage  of 
pure  substance  contained  in  the  mixture  be  known.  It  is  hardly 
necessary  to  say  that  these  principles  should,  as  far  as  possible,  be 
applied  to  the  study  of  toxins.  In  these  substances  also  one  should 
know  the  degree  of  purity  before  attempting  any  exact  investigations 
Lut  just  in  this  domain,  where  it  is  impossible  to  isolate  the  substances, 
this  task  is  uncommonly  difficult.  It  required  a  year's  most  tiresome 
and  monotonous  labor  before  I  was  able,  by  means  of  very  exact  deter- 
minations of  all  kinds  of  poisons,  to  approach  this  problem.  At  that 


produced  in  a  very  short  time  (three  to  four  days  in  the  incubator)  are  not  free  from 
toxoids.  In  one  such  poison  (No.  9  of  the  titration  series)  I  found  123  L.  D. 
in  Lt.  I  was  therefore  greatly  pleased  recently  to  hear  from  Dr.  Louis  Martin, 
who  has  had  such  wide  experiences  in  this  direction  at  the  Pasteur  Institute, 
that  in  his  fresh  poisons  he  never  saw  the  figure  200  L.  D.  in  Lf  reached. 


490  COLLECTED  STUDIES  IN  IMMUNITY 

time  I  gained  the  impression  that  a  pure  poison  must  oe  so  consti- 
tuted that  one  I.  E.  fully  neutralizes  exactly  200  L.  D.1  Later  on  I 
shall  show  that  by  means  of  the  "spectrum"  analysis  I  have  suc- 
ceeded in  verifying  this  figure.2 

The  discovery  of  this  number,  200,  led  me  to  represent  the  con- 
stitution of  diphtheria  poison  by  means  of  a  " spectrum"  which  is 
divided  into  200  segments,  each  of  which  corresponds  to  a  toxin, 
toxoid,  or  toxon  equivalent.  This  scheme  is  not,  as  some  have  as- 
sumed, a  mere  makeshift,  but  is  the  expression  of  knowledge  labori- 
ously attained.  This  graphic  reproduction  shows  at  a  glance  how 
much  toxin  or  toxoid  is  neutralized  by  each  combining  unit  of  anti- 
toxin. Such  a  reproduction  possesses  so  many  advantages  over  the 
curve  used  by  Arrhenius  and  Madsen  that  I  shall  not  hesitate  a  moment 
in  retaining  the  spectrum  method  for  diphtheria  poison.  By  its 
means  one  obtains  a  view  of  the  entire  process  of  neutralization.3 

It  may  be  well  at  this  point,  by  means  of  a  suitable  chemical 
illustration,  to  elucidate  the  influence  which  such  admixtures  of 
toxoid  exert  in  the  titration  of  alkaloids.  In  doing  this  it  will  be 
best  to  proceed  on  the  following  assumptions.  An  alkaloid  acts  hsemo- 
lytically  when  in  the  form  of  free  base,  but  not  when  in  the  form  of 
a  salt.4  The  base  would  then  correspond  to  the  toxin.  The  ana- 
logue of  the  toxoid  would  then  be  an  alkaloid  which  exerts  no  dele- 
terious action  either  as  such  or  in  the  form  of  a  salt.  The  antitoxin 
would  be  represented  by  any  acid,  e.g.,  hydrochloric  acid.  Under 
these  conditions  the  mixture  of  the  two  alkaloids  can  be  titrated  bio- 
logically (by  determining  the  haemolytic  power  at  any  point)  by 
means  of  an  acid  exactly  as  a  toxin  solution  containing  toxoid  by 
means  of  its  antitoxin. 

Let  us  assume  that  the  toxic  alkaloid  A  as  well  as  the  atoxic  B 
possesses  so  strong  an  affinity  for  hydrochloric  acid  that  neutraliza- 
tion is  effected  to  within  a  very  small  fraction.  A  solution  of  a  mole- 
cules A  would  then  correspond  to  the  pure  toxin,  while  mixtures  of 

1  It  is  self-evident  that  each  toxin-combining  unit  can  be  replaced  by  an 
equivalent  amount  of  less  toxic  or  non-toxic  substances  possessing  combining 
properties  (toxones,  toxoids). 

'  The  poison  studied  by  Madsen,  therefore,  which  contained  160  L.  D.  in 
Lf,  corresponded  to  a  purity  of  four-fifths. 

3  See  also  page  552. 

4  This  is  probably  the  case  with  solanin,  whose  hsemolytic  power  is  inhibited 
by  the  addition  of  acid  salts  (Pohl)  or  ot  free  acids  (He"don,  Bashford). 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  491 

A  and  B:  ?+^  or  J+-T-  represent  analogues  of  solutions  containing 

also  toxoids.  In  all  of  these  mixtures  the  end  point  of  neutralization 
will  be  practically  constant.  If,  however,  the  affinities  of  A  and  B 
for  hydrochloric  acid  are  not  exactly  equal  the  neutralization  will 
proceed  in  a  straight  line  only  if  we  are  dealing  with  the  pure  alkaloid. 
In  all  other  cases  it  will  follow  the  course  of  a  curve  whose  character, 
of  course,  is  dependent  on  the  relative  amounts  of  the  two  com- 
ponents. 

This  problem  of  the  simultaneous  neutralization  of  two  alkaloids 
has  been  studied  in  suitable  cases  by  J.  H.  Jellet.  Let  us  take  the 
neutralization  of  quinine  and  codein  with  hydrochloric  acid,  in  which 
the  coefficient  of  equilibrium  /£  =  2.03.  For  the  sake  of  simplicity 
I  have  assumed  this  to  be  2.0.  In  order,  furthermore,  to  have  the 
conditions  as  simple  as  possible,  let  us  take  as  an  example  a  mixture 
of  100  molecules  quinine  and  100  molecules  codein.  These  will  then 
be  neutralized  by  200  molecules  hydrochloric  acid.  By  means  of 
the  formula  devised  by  Jellet  one  next  determines  how  much  quinine 
is  transformed  into  the  salt  by  each  successive  addition  of  one-tenth 
the  entire  neutralizing  dose  (20  molecules  HC1).  It  will  be  found 
that  the  first  tenth  neutralizes  13  and  the  last  tenth  7  molecules 
of  quinine,  while  the  course  of  the  neutralization  of  the  quinine  is 
itselt  entirely  uniform.  If  another  combination  is  taken,  in  which 
the  second  alkaloid  possesses  a  weaker  affinity,  so  that  K  =  1Q,  it  can 
easily  be  calculated  that  under  these  circumstances  the  first  tenth 
hydrochloric  acid  neutralizes  17.8,  the  last  tenth  only  3  molecules 
of  quinine.  On  representing  these  reactions  graphically  we  shall 
obtain  curves  entirely  similar  to  those  representing  the  neutralization 
of  a  weak  base  with  a  weak  acid,  and  it  would  probably  not  be  difficult 
to  find  a  combination  of  alkali  and  acid  whose  curve  corresponds  to 
the  alkaloid  curve  mentioned. 

Hence,  if  such  a  mixture  of  alkaloids  together  with  the  appro- 
priate neutralizing  agent  (acid)  were  given  one  for  a  biological  titra- 
tion,  and  if,  furthermore  (to  make  the  analogy  with  toxin-antitoxin 
determination  complete),  the  employment  of  any  additional  chemical 
aids  was  barred,  the  neutralization  curve  obtained  under  such  stringent 
conditions  could  easily  give  the  impression  that  one  were  dealing 
only  with  the  neutralization  of  two  substances  possessing  weak  affini- 
ties. Nevertheless,  even  under  these  limitations,  it  is  possible  to 
learn  the  true  conditions  if,  as  I  have  done,  one  does  not  confine  one's 


492  COLLECTED  STUDIES  IN  IMMUNITY. 

self  to  a  single  mixture,  buju  analyzes  a  great  many  different  mixtures 
in  which  the  relation  of  toxin-alkaloid  and  toxoid-alkaloid  varies.1 

It  is  all  the  more  surprising  that  in  the  analysis  of  the  constitu- 
tion of  poisons  Arrhenius  and  Madsen  have  not  studied  the  question 
from  this  point  of  view  because  they  do  not  at  all  neglect  the  exist- 
ence of  toxoids.  Apparently  this  is  because  of  a  slight  misunder- 
standing, for  these  authors  proceed  exclusively  on  the  assumption  that 
in  toxoids  one  is  dealing  with  protoxoids,  i.e.,  with  toxoids  which 
possess  a  higher  affinity  for  the  antitoxin  than  does  the  toxin.  In 
fact,  one  can  easily  observe  that  the  formation  of  prototoxoids  affects 
the  end  point  of  the  titration  but  little.  This  I  had  predicted  in  my 
first  study  on  the  evaluation  of  diphtheria  serum.  Let  us  assume, 
for  example,  that  a  mixture  of  1  equivalent  hydrochloric  acid  (proto- 
toxoid)  and  3  equivalents  prussic  acid  (toxin)  is  neutralized  by  a 
strong  base.  In  that  case  the  hydrochloric  acid  will  be  neutralized 
first,  after  which  the  neutralization  of  the  prussic  acid  will  proceed 
very  much  the  same  as  though  only  prussic  acid  were  present. 

We  must  now  see  whether  diphtheria  poisons,  such  as  I  have 
investigated,  contain  other  toxoids  besides  prototoxoids.  The  ma- 
terial at  hand  makes  the  decision  of  this  point  very  simple.  In  four 
poisons  containing  a  prototoxoid  zone  (of  which  two  were  published 
by  myself  and  two  by  Madsen)  I  have  calculated  the  relation  of  proto- 
toxoid and  toxoid  to  toxin.  In  doing  this  I  have  regarded  exclusively 
the  LI  dose,  and  so  eliminated  the  toxons  which  would  otherwise  still 
more  increase  the  toxoid  figure. 


1  In  the  very  simple  example  of  two  alkaloids  just  mentioned  two  determina- 
tions of  different  mixtures  would  permit  the  calculation.  In  my  opinion  no 
definite  conclusions  as  to  the  constants  of  the  toxin  can  be  drawn  from  the 
analysis  of  one  particular  toxin  containing  toxoid.  Arrhenius  and  Madsen 
analyzed  two  different  tetanus  poisons,  one  of  which  had  undergone  toxoid 
modifications  through  years  ot  preservation  as  a  dry  substance,  while  the  other 
had  suffered  similar  modifications  through  several  days'  standing  of  the  solu- 
tion. The  authors  calculated  from  their  experiments  that  in  the  one  case  the 
constant  of  dissociation  had  been  increased  50%,  in  the  other  ten  times.  In 
view  of  what  has  just  been  stated  this  calculation,  which  leaves  out  of  account 
the  presence  of  toxoids,  cannot  be  regarded  as  conclusive.  The  divergence  of 
the  constants  could  easily  be  due  exclusively  to  the  presence  of  toxoids,  and 
these,  in  view  of  the  different  methods  by  which  the  poisons  were  attenuated, 
could  be  different  in  the  two  cases.  I  may  also  add  that  in  the  toxoid  for- 
mation of  diphtheria  toxins  I  am  convinced  that  the  toxin  groups  which 
remain  do  not  suffer  any  change  in  their  affinity. 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN. 


493 


Poison. 

For  100  Parts  of  Toxin  there  are 

Prototoxoid, 
Parts 

Toxoid. 
Parts. 

A     Mad  sen 
C     Madsen 
IV  Ehrlich 
V     Ehrlich  (4th  phase) 

160 
79 

82 

77 

400 
59 
200 
131 

This  table  shows  that  the  four  poisons  contain  considerable  amounts 
of  toxoids  in  addition  to  the  prototoxoids.  The  affinity  of  these 
toxoids  is  more  or  less  small,  as  can  be  seen  from  the  curves  plotted 
by  Madsen  and  myself.  From  this  it  follows  that  in  the  interpreta- 
tion of  the  results  obtained  by  neutralizing  diphtheria  poison  due 
attention  must  be  paid  to  the  decisive  influence  exerted  on  the  course 
of  the  partial  neutralization  by  the  toxoids  notoriously  present  in  such 
considerable  amounts.  It  is  incorrect,  therefore,  to  refer  the  decreased 
binding  of  antitoxin,  such  as  is  seen  in  the  tritotoxoid  zone,  to  the 
boric  acid-ammonia  scheme. 

It  will  be  well,  by  means  of  a  concrete  example,  to  study  some- 
what more  in  detail  the  course  of  this  toxoid  formation.  For  this 
purpose  1  shall  select  a  poison  wrhich  I  have  already  described  in  my 
publication  on  the  constitution  of  diphtheria  poison  l  as  Poison  No.  5. 
At  that  time  I  briefly  gave  the  spectrum  and  the  constants  based  on 
the  investigations  which  I  and  my  friend  Donitz  had  carried  out.  In 
this  poison  the  conditions  were  most  interesting  and  yet  extremely 
simple:  The  L0  dose  was  0.125  cc.;  the  Lf  dose  0.25  cc.,  that  is,  just 
twice  as  much.  The  L.  D.  was  0.0025  cc., so  that  the  LQ  dose  contained 
exactly  50  L.  D.  and  the  Lt  dose  exactly  100  L.  D.  These  facts 
caused  us  to  make  the  thorough  analysis.  This  poison,  as  is  so  often 
the  case,  suffered  certain  transformations,  whereby  it  became  weaker. 
These  changes  occurred  in  three  phases  characterized  by  the  formation 
of  different  kinds  of  toxoids.  The  spectra  of  these  phases  are  as  fol- 
lows (Fig.  1). 

The  phases  in  which  the  content  of  toxin  shows  itself  are  I,  II,  and 
IV;  phase  III,  which  deals  with  the  toxons,  will  be  considered  in  a 
separate  chapter. 

As  a  result  of  all  my  experiences  with  similar  poisons,  as  well  as 


'  Deutsche  med.  Wochensch.  1898,  No.  38. 


494 


COLLECTED  STUDIES  IN    IMMUNITY. 


from  a  direct  determination,  it  follows  that  the  first  phase  must  have 
represented  a  pure  hemitoxin  which  reached  exactly  to  100  (see  illus- 
tration). Accordingly  each  r—  I.  E.  (  =  1  combining  unit)  succes- 
sively added  to  the  L  dose  takes  away  J  L.  D.  from  the  fatal  doses 
Phase  I 


0   10  20  30  40  50  60  70 


90  100  110  120  130  140  150  160  170  180  190  2CO 


Phase  II 


10 


10  .20  30  40  .50  00   70  80  90  100  110  120  130  140  150  160  170  180  190  200' 

Phase  III 


10 


0   10  20   30  40   50  60  70  80  90  100  110  120  130  140  150  160  170  180  190  200- 

Phase  IV 


0   10  20  30  40  50  00  70  80  90  100  110  120  130  140  150  160  170  180  190  200 

FIG    1 

contained  inL0,  and  this  all  occurs  within  the  first  hundred 
antitoxin  doses  added.  Amounts  of  antitoxin  beyond  this  have  no 
further  influence  on  the  toxin  (death,  necrosis),  but  affect  only  the 
toxon. 

A  fact  to  which  I  attach  particular  significance  is  that  the  hemi- 


THE  CONSTITUENTS   OF  DIPHTHERIA   TOXIN  495 

toxin  reaches  just  up  to  the  100  limit  and  shows  no  trace  of  any 
gradual  decline.  This  follows  from  the  determination  of  the  Lt  dose, 
as  can  be  seen  from  the  following  analysis. 

Given  a  poison  in  which,  in  the  L0  dose,  the  hemitoxin  zone  reaches. 
exactly  to  100,  how  large  will  the  Lt  dose  be?  Lt,  i.e.,  the  amount 
of  poison  which  on  the  addition  of  200  combining  units  still  leaves 
1  L.  D.  free,  will  be  reached  when  200  equivalents  of  hemitoxin  are 
present.  We  shall  therefore  have  to  multiply  the  L0  dose  of  the 

202 
poison  by  -  -  in  order  to  obtain  the  Lt  dose.     If  we  carry  out  this 

1  \J\J 

multiplication  we  obtain  an  Lt  dose  of  0.253,  which  agrees  very  well 
with  the  value  actually  found,  0.25  cc. 

Thus  the  important  fact  is  demonstrated  that  in  this  case  the 
neutralization  of  the  diphtheria  poison  by  antitoxin  proceeded  exactly 
the  same  as  the  neutralization  of  a  strong  acid  by  a  strong  base. 
Here  then  the  course  of  the  reaction  is  represented  by  a  straight  line 
and  not  by  a  curve. 

Further  evidence  for  the  view  that  in  this  poison  the  hemitoxin 
extended  right  up  to  the  limit  100  is  furnished  by  phase  II.  Here 
we  see  a  simultaneous  increase  of  the  Lt  dose  and  a  decrease  of  the 
toxidty  manifesting  themselves  by  the  fact  that  the  L.  D.  increases 
from  0.0025  to  0.003  cc.,  so  that  the  number  of  L.  D.  contained  in  the 
L0  dose  has  decreased  from  50  to  42. 

This  increase  cf  the  Lt  dose  amounted  to  about  0.26  cc.  and  from 
it,  by  means  of  the  simple  calculation  already  mentioned,  it  can  be 
shown  that  toxoid  formation  took  place  in  the  end  zone  of  the  toxin, 
the  "tritotoxoid  zone,"  as  I  term  it. 

Let  us  assume  that  the  end  zone  (which  before  as  well  as  after  the 

second  phase  extended  to  100)  contains  a  toxoid  mixture  of  —  toxicity 

instead  of  the  hemitoxin.      In  order  to  reach  the  Lt  dose  in  .this 

210  20^ 

case  we  must  multiply  the  L0  dose  by  —  -  and  not  by  -  —  ,  as  was 

.ZUU  '  zOO 

the  case  with  hemitoxin.  On  carrying  out  this  calculation,  L0  being 


In  the  determination  made  at  that  time  I  actually  found  the  Lt 
dose  to  be  0.26,  but  noted  "a  little  over."     That  the  tritotoxoid  zone 

possessed  a  toxicity  of  —  was  shown  by  the  subsequent  analysis  by 
means  of  partial   neutralization,  for  near  the  end,  a  zone  of  18-20 


496  COLLECTED  STUDIES  IN   IMMUNITY. 

tritotoxid  of  exactly  —  toxicity  was  found.     It  should  be  emphasized 

that  the  fatal  doses  which  disappeared  in  the  deterioration  were  found 
in  the  form  of  toxoids  in  the  tritotoxoid  zone. 

These  investigations  show  that  these  changes  are  due  exclusively  to 
the  fact  that  a  part  of  the  toxin  has  become  transformed  into  toxoids ; 
in  fact  into  toxoids  which  are  neutralized  after  the  main  portion  of 
the  toxin,  and  which,  therefore,  must  possess  less  affinity.  If  we  were 
to  represent  this  phase  by  means  of  a  curve  according  to  the  method 
of  Arrhenius  and  Madsen,  we  should  observe  a  marked  flattening 
of  the  curve  in  the  tritotoxoid  zone.  This,  however,  is  not  the  expres- 
sion of  the  weak  affinity  of  the  diphtheria  toxin,  or  of  the  neutraliza- 
tion dependent  thereon.  It  is  to  be  ascribed  with  absolute  certainty 
solely  to  the  presence  of  toxoids  and  their  appearance  in  place  of  toxin 
molecules  which  have  disappeared. 

I  shall  discuss  phase  III  later,  merely  remarking  at  this  time  that 
in  this  phase,  80  out  of  100  parts  toxon  have  disappeared.  The  L0 
dose  of  0.125  cc.  now  contains  only  120  combining  units  instead  of 
the  200  units  (toxin  and  toxon)  originally  present.  Corresponding 
to  this,  therefore,  the  L0  dose,  which  must  contain  200  combining  units, 
increases  from  0.125  cc.  to  0.21  cc.  In  this  third  phase  the  toxin 
zone  has  not  suffered  any  essential  change.  The  L-j-  dose  has  accord- 
ingly remained  constant  at  0.26  cc.  Because  of  the  new  L0  dose  made 
necessary  by  the  loss  of  toxon,  the  spectrum  representing  this  phase 
shows  a  much  wider  toxin  zone  than  the  previous  one.  The  toxin- 
toxon  boundary  has  been  moved  from  100  to  166.  - 

In  phase  IV,  L-j-  remained  0.26  cc.,  but  the  toxicity  decreased, 
the  L.  D.  increasing  gradually  from  0.003  cc.  to  0.004  cc.  During  the 
course  of  these  changes  22  L.  D.  had  disappeared  from  the  L0  dose  of 
phase  III. 

The  fate  of  these  22  L.  D.  is  made  plain  by  the  spectrum  which 
I  constructed  at  that  time.  In  this  I  found  an  extended  prototoxoid 
zone  which  included  the  first  40  combining  units  of  the  spectrum, 
sufficient,  as  can  be  seen,  to  explain  the  loss  of  toxin  which  had  oc- 
curred. I  desire  to  call  particular  attention  to  the  fact  that  no  loss 
of  combining  groups  had  occurred  despite  the  slight  increase  of  the 
L  dose.1 


1  A  superficial  glance  might  lead  one  to  suppose  that  the  fact  that  the  Lf 
dose  of  0.25  cc.  in  the  first  phase  had  become  increased  to  a  little  over  0.26  cc., 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN,  497 

This  behavior  shows  that  on  standing  there  is  not,  for  example, 
a  marked  destruction  of  the  poison,  but  merely  a  slight  chemical 
change  affecting  only  the  toxophore  and  not  the  haptophore  group. 
It  would  be  improper,  therefore,  to  speak  of  the  poison  "spoiling." 

The  observations  on  the  origin  in  the  various  forms  of  toxoid  are 
particularly  important. 

In  the  first  phase  of  toxin  formation,  there  was  a  development 
of  toxoids  of  weaker  affinity  for  the  antitoxin,  while  during  the  second 
stage,  toxoids  of  greater  affinity  developed.  Occupying  a  position 
between  these  two  opposing  poison  modifications  is  the  hemitoxin 
fraction,  and  this  has  remained  intact.  We  are  thus  really  forced  to 
arrange  these  three  poison  constituents,  according  to  their  affinity, 
as  prototoxoid,  deutero  toxoid,  and  tri  to  toxoid.  This  brings  me  to 
the  crux  of  my  views  concerning  the  constitution  of  diphtheria  poison. 

In  titrating  and  evaluating  the  diphtheria  antitoxic' serum  I  began 
with  the  simplest  assumption,  namely,  that  the  poison  was  a  simple 
uniform  substance.  In  the  formation  of  toxoids,  therefore,  I  con- 
sidered three  possibilities: 

1.  That  the  affinity  of  the  haptophore  becomes  increased; 

2.  That  it  remains  the  same,  and 

3.  That  it  decreases. 

Which  of  these  possibilities  will  apply  in  any  given  case  will,  of 
course,  depend  upon  the  stereochemical  circumstances,  especially 
upon  how  far  one  functionating  group  is  removed  from  the  other. 
If,  in  what  we  must  conceive  to  be  a  very  large  molecule,  these  groups 
are  quite  far  apart,  it  may  be  assumed  a  priori  that  the  destruction  of 
the  toxophore  group  will  probably  not  exert  a  marked  influence  on 
the  haptophore  group.  In  other  words,  syn toxoids  will  be  formed. 
If  the  two  groups  are  nearer  together  a  change  in  the  affinities,  either 
positively  or  negatively,  can  readily  occur.  As  a  matter  of  fact, 
the  possibility  of  an  increase  or  decrease  of  affinity  as  a  result  of  this 
transformation  into  inert  modifications  has  also  been  observed  in  con- 
nection with  related  subjects.  Researches  conducted  by  myself  and 
Sachs  have  shown  that  in  the  formation  cf  complementoid  the  hap- 

was  the  expression  of  a  certain  loss  of  combining  groups.  This,  however,  is 
merely  apparent;  in  the  second  phase  a  greater  excess  of  the  poison  (containing, 
as  it  does,  more  toxoid)  is  required  to  produce  death  than  is  the  case  with 
the  haemitoxin.  Bearing  this  consideration  in  mind  it  is  easy  to  convince 
one's  self  that  not  a  single  one  of  the  combining  groups  present  has  been  lost 
and  that  the  change  which  the  poison  has  undergone  was  a  quantitative  one. 


498 


COLLECTED  STUDIES  IN  IMMUNITY 


tophore  group  suffers  a  decrease  in  affinity.  Complementoids,  it  will 
be  remembered,  result  from  the  destruction  of  the  zymotoxic  group, 
the  analogue  of  the  toxophore  group.  Eisenberg  and  Volk  by  their 
discovery  of  proagglutinoids  have  shown  that  in  the  formation  of 
agglutinoids  an  increase  in  affinity  can  take  place. 

Hence  in  diphtheria  poison  the  possibility  had  to  be  considered 
that  similar  conditions  obtain  in  toxoid  transformation.  In  this  case, 
however,  it  was  remarkable  that  this  toxoid  formation  did  not  always 
follow  the  same  scheme,  the  poison,  of  course,  always  being  thought 
of  as  a  simple  uniform  substance.  I  was  finally  able  to  solve  this 
problem  in  the  following  manner. 

My  earlier  investigations  had  given  me  the  impression  that  1  I.  E. 
(immune  unit)  should  neutralize  200  fatal  doses  of  a  pure  toxin,  one 
consisting  only  of  toxin  molecules  and  therefore  free  from  toxoids. 
I  am  quite  ready  to  admit  that  I  did  not  at  that  time  furnish  any 
absolute  proof  for  this  view.  My  first  effort  was  therefore  directed 
to  a  study  concerning  the  correctness  of  the  figure  200.  I  began  by 
analyzing  a  large  number  of  different  toxins  in  the  hope  that  sooner 
or  later  I  would  find  an  ideally  pure  toxin.  I  have  already  men- 
tioned that  the  highest  purity  thus  far  obtained,  a  toxin  obtained 
by  Madsen,  corresponds  to  only  four-fifths  purity,  L-f-  containing  160 
L.  D.  Nevertheless  by  means  of  the  method  of  neutralization  I  was 
able  to  find  poisons  which  fulfilled  my  requirements,  at  least  in  part. 
This  was  the  case,  for  example,  in  my  Poison  No.  2  (see  spectrum, 
Fig.  2).  In  this  the  L0  dose  contained  84  L.  D.  The  first  third  of  the 


10  20  30  40  60  60 


80  90  100  110  120  130  140  150  .160  .170  .180  190  200 

FIG.  2. 


spectrum  was  taken  up  by  a  zone  of  hemitoxin  not  quite  pure.,  i.e.,  each 
combining  unit  added  (  —  I-  E.  J  decreased  the  toxicity  by  about  - 

L.  D.  In  the  next  zone,  on  the  other  hand,  stretching  from  72  to  115, 
each  combining  unit  took  away  exactly  1  L.  D.  The  spectrum  is 
here  reproduced.  It  shows  the  zones  of  hemitoxin,  pure  toxin,  trito- 
toxoid,  and  toxon  very  clearly. 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  499 

Madsen,  too,  has  described  a  poison  "C,"  the  constitution  of 
which  is  very  interesting  because  prototoxoid  and  pure  toxin  are 
distinctly  marked  off  from  one  another.  During  the  phase  at  which 
Madsen  examined  it  the  pure  toxin  zone  occupied  the  zone  50  to  100 
of  the  spectrum.  Before  the  formation  of  tritotoxoid  this  zone  may, 
however,  have  extended  to  150. 

From  these  observations  we  see  that  for  certain  portions  of  the 
spectrum  (which  lie  in  the  middle  and  not  at  the  commencement  1) 

it  has  been  possible  to  prove  that  -p^-z.  I.  E.  combines  with  exactly 


1  L.  D.  This  argues  strongly  in  favor  of  the  correctness  of  my 
assumed  figure  200.  In  these  zones  of  pure  toxin  only  toxin  molecules 
are  neutralized  and  no  toxoids. 

Although  it  is  rare  to  find  zones  of  pure  toxin  in  poisons  which  have 
been  kept  some  time,  it  is  extremely  common,  or  even  constant,  to- 

find  in  these  older  poisons  zones  in  which  —=:  I.  E.  neutralizes  exactly 

J  L.  D.  Manifestly  under  these  conditions  equal  parts  of  toxin  and 
toxoid  must  always  be  neutralized;  for  this  reason  I  have  termed 
such  a  poison  a  hemi  toxin.  The  following  scheme  represents  such  a 
changed  poison: 


T 


TT 


Toxin :  Pure  Toxin 


Toxoid:  Hemitoxin 


FIG,  3. 


It  needs  no  further  explanation  to  show  that  in  this  hemitoxin 
zone  the  affinity  of  toxin  and  toxoid  to  antitoxin  has  remained  un- 
changed. 

The  entire  process  of  toxoid  formation  takes  place  in  two  phases, 
as  can  readily  be  seen  from  the  initial  zones  of  suitable  spectra  (see 
Fig.  3).  The  pure  toxin  first  changes  into  hemitoxin;  in  the  second 
phase,  however,  the  hemitoxin  changes  into  pure  toxoid,  especially 
in  the  first  part  of  the  spectrum.  This  is  illustrated  by  the  following 
scheme : 

1  In  the  curve  of  ammonia-boric  acid  and  of  tetanolysin  the  maximum 
combining  power  always  occupies  the  very  first  portions  of  the  curve. 


500  COLLECTED   STUDIES  IN  IMMUNITY. 

/\  I   :  Pure  Toxin 

Toxin  / 


V 


T 


Toxoid. 


:  Hemitoxin  (Hemi- 
toxoid). 


:  Pure  Toxin 


FIG.  4. 


I  must  again  emphasize  that  this  sketch  of  the  decomposition 
of  the  poison  is  not  at  all  hypothetical,  but  merely  the  expression 
of  the  facts  observed.  The  regular  course  in  two  phases  points  di- 
rectly to  the  fact  that  the  individual  toxins  are  not  simple  uniform 
substances  but  are  composed  of  two  modifications  present  in  equal 
amounts  in  the  toxin  solution  and  behaving  differently  on  decompo- 
sition. One,  the  more  unstable  of  the  two,  the  ct-modification,  decom- 
poses rapidly  and  so  gives  rise  to  the  stage  of  hemitoxin.  The  subse- 
quent destruction  of  the  more  stable  /^-modification  leads  to  pure 
toxoid.  It  is,  of  course,  somewhat  remarkable  that  exactly  equal 
parts  of  two  toxin  modifications  should  develop  in  diphtheria  bouillon. 
This  is  readily  understood,  however,  if  we  remember  that  E.  Fischer 
has  made  it  extremely  probable  that  the  active  groups  of  ferments 
(groups  exhibiting  a  great  similarity  with  the  toxophore  group)  pos- 
sess an  asymmetrical  constitution.  If  then  in  accordance  with  this 
we  assume  an  asymmetrical  constitution  of  the  toxophore  group,  there 
will  be  nothing  remarkable  in  the  fact  that  the  diphtheria  bacilli 
produce  both  asymmetrical  components  simultaneously.  Nor  is  it 
surprising  that  both  are  produced  in  equal  amounts  if  we  consider, 
for  example,  that  optically  inactive  tartaric  acid  consists  of  equal 
parts  of  dextro  and  Isevo  tartaric  acid.  If  optically  active  combina- 
tions (of  which  a  large  number  can  be  made  artificially)  are  produced 
in  the  retort,  the  rule  holds  that  exactly  the  same  number  of  mole- 
cules of  the  two  components  are  produced  by  the  reaction. 

Ever  since  Pasteur  showed  that  in  the  fermentation  of  tartaric 
acid  by  moulds  the  dextro  tartaric  acid  is  decomposed  first,  it  has  been 
found  possible  to  demonstrate  a  similar  behavior  in  numerous  other 
instances ;  thus  by  the  aid  of  moulds,  yeasts,  and  bacteria  it  was  found 
possible  to  isolate  one  of  the  optically  active  components  from  racemic 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN 


501 


combinations.  Looked  at  in  this  way  the  formation  of  hemitoxin  is 
explained  in  very  simple  fashion.1 

It  can  readily  be  shown  that  in  the  first  stage  of  toxoid  formation 
which  leads  to  hemitoxin  no  change  in  affinity  takes  place,  and  this 
holds  true  also  for  all  the  toxoid  formation,  for  if  an  increase  in 
affinity  occurred  there  could  be  no  hemitoxin  zone ;  a  prototoxoid 
zone  would  again  be  followed  by  a  zone  of  pure  toxin.  Conversely 
if  there  were  a  decrease  in  affinity  a  zone  of  pure  toxin  would  precede 
the  toxoid  portion.  The  following  scheme  will  serve  to  make  these 
conditions  clear: 

These  considerations 
at  once  show  us  that  in 
the  formation  of  toxoid 
no  change  in  affinity  can 
take  place.  As  a  matter 
of  fact,  however,  the  pro- 
totoxoid possesses  a  much 
stronger,  and  the  trito- 
toxoid  a  much  weaker, 
affinity  than  the  toxin  or 
hemitoxin  occupying  the 
central  portion  of  the 

spectrum.  This  we  saw  in  our  analysis  of  the  poison  mentioned 
above.  We  must,  therefore,  conclude  that  this  difference  is  not 
produced  by  the  formation  of  toxoid,  but  exists  in  the  toxic  bouil- 
lon from  the  beginning,  the  initial  portion  of  toxin,  which  subse- 
quently passes  over  into  prototoxoid,  already  possessing  a  higher 
affinity  for  the  antitoxin.  The  poison  of  diphtheria,  for  example, 
could  be  represented  by  the  following  rough  diagram,  in  which  the 
degree  of  affinity  is  expressed  schematically  by  the  length  of  the  lines : 


TTTT 


i        i 

Tl 
T  I 


FIG    5. 


Pure  Toxin 


Increased 
Affinity 


Decreased 
Affinity 


Affinity 
Unchanged 


-    i  in*- 

—xx  —  • 

•^111 

m 
s          ^ 

m           im 
n  ^» 

iM            • 
-~^-  

—            — 

~^ 

• 

;.U 

JJ 

Erototoxin                         Deuterotoxin                          Tritotoxin 

FIG.  6. 

1  See  E.  Fischer.  Zeitschr.  f.  physiol.  Chemie,  Vol.  26. 


502  COLLECTED  STUDIES  IN   IMMUNITY. 

Certain  other  considerations  have  convinced  me  of  the  plurality 
of  the  toxins.  Chief  of  these  is  the  behavior  of  the  poisons  on  long 
standing.  As  is  well  known,  poisons  freshly  produced  rapidly  deterio- 
rate in  toxicity  until  a  point  is  reached  beyond  which  the  constants 
of  titration,  especially  Lj,  remain  unchanged.  Such  "  ripened  "  poisons 
are  made  use  of  in  the  official  testing  of  diphtheria  antitoxin,  and  we 
have  therefore  had  abundant  opportunity  to  convince  ourselves  that 
they  remain  constant. 

From  the  standpoint  of  physical  chemistry  this  fact  (that  the 
toxicity  after  a  time  becomes  constant)  could  perhaps  be  ascribed 
to  an  equilibrium  between  toxin  and  toxoid.  Such  an  equilibrium, 
however,  is  found  only  in  reversible  reactions,  i.e.,  in  chemical  proc- 
esses, which  also  proceed  in  the  reverse  direction.  Toxoid  formation, 
however,  is  not  a  reversible  reaction ;  no  one  has  yet  discovered  even 
a  suggestion  of  a  toxoid  passing  over  into  toxin.  Another  point  which 
speaks  against  a  condition  of  equilibrium  is  the  fact  that  through 
artificial  influences— heat,  chemicals — any  desired  proportion  of  toxin 
and  toxoid  can  be  produced.  Only  one  other  explanation  therefore 
remains,  namely,  that  various  toxins  are  present,  of  which  some  are 
more  resistant,  others  less  so. 

I  have  thus  presented  in  detail  the  reasons  which  led  me  to  assume 
the  existence  of  preformed  varieties  of  toxins.  As  a  result  of  my  ex- 
periments I  must  emphatically  deny  the  assumption  that  the  phe- 
nomena observed  by  me  in  diphtheria  poison  are  only  the  expression 
of  a  weak  affinity  between  diphtheria  toxin  and  antitoxin.  I  have 
demonstrated  that  the  observed  deviations  can  only  be  due  to  the 
admixture  of  toxoids  with  different  affinity,  and  have  further  made 
it  probable  that  these  different  degrees  of  affinity  exist  preformed 
in  the  toxin  and  do  not  arise  with  the  formation  of  toxoid.  It  must, 
however,  be  distinctly  understood  that  the  points  of  view  here  laid 
down  are  not  applicable  to  the  relations  between  toxins  and  antitoxins 
in  general.  They  apply  only  to  diphtheria  toxin  and  its  antitoxin. 
The  important  researches  of  Arrhenius  and  Madsen  on  tetanolysin 
show  that  neutralization  proceeds  in  an  entirely  different  fashion 
when  the  two  components  possess  a  weak  affinity  for  one  another. 
The  studies  of  these  authors  clearly  indicate  the  errors  in  the  interpre- 
tation of  neutralization  phenomena  when  dissociation  is  disregarded. 

My  results  were  obtained  by  the  long  and  tedious  experimental 
method.  I  can  assure  the  reader  that  the  experiments  upon  which 
all  this  is  based,  experiments  carried  out  by  my  fellow  workers  (espe- 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  503 

cially  Geh.-Rath  Donitz  and  Dr.  Morgenroth)  and  myself,  have  been 
most  exact,  and  I  venture  to  say  that  in  medicine  but  few  investiga- 
tions exist  which  have  been  carried  out  with  such  precision  and  on 
such  abundant  material. 

II.  Toxons. 

Thus  far  we  have  dealt  only  with  the  true  toxin  portion  of  the 
diphtheria  poison,  and  have  entirely  disregarded  another  constant 
secretory  product  of  the  diphtheria  bacillus,  namely,  the  toxons.  On 
testing  a  diphtheria  poison  and  determining  the  two  limits,  L0  and  LJ, 
we  should  expect  that  the  difference,  L-J--L  =D,  would  correspond 
exactly  to  one  lethal  dose,  provided  the  poison  were  a  simple  uniform 
substance.  Thus  if  L  ,  for  example,  contains  a  lethal  doses  these, 
according  to  our  definition  of  LQ,  will  exactly  be  neutralized  by 
1  I.  E.  Assuming  that  the  two  substances  have  a  strong  affinity  for 
each  other,  the  addition  of  one  L.  D.  would  suffice  to  transform  this 
neutral  L0  mixture  into  Lf,  i.e.,  Lt  should  contain  (a+1)  L.  D.  and  the 
difference,  D,  should  equal  1.  As  a  matter  of  fact,  however,  it  was 
found  that  with  the  exception  of  one  poison  examined  by  me,  the 
difference  between  Lt  and  L0  is  much  greater.  In  the  poisons  de- 
scribed in  my  first  communications  the  difference  D  ranged  from 
5  to  50  L.  D.  At  first,  when  I  still  held  to  the  Unitarian  conception, 
I  had  interpreted  these  results  as  indicating  the  existence  of  a  toxin 
derivative  of  very  little  toxicity  and  possessing  less  affinity  than  the 
toxin.  For  this  reason  I  termed  the  derivative  "epitoxoid."  In  my 
second  communication,  however,  I  abandoned  this  assumption,  and 
stated  that  we  were  evidently  dealing  with  a  primary  secretory  prod- 
uct of  the  diphtheria  bacilli— the  "toxon."  The  reasons  which  led 
me  to  this  view  will  be  presented  in  a  moment.  The  toxon  possesses 
the  same  haptophore  group  as  the  toxin,  but  a  weaker  affinity  for  the 
antitoxin.  The  main  difference  is  in  the  toxophore  group,  for  even 
when  given  in  large  doses  the  toxon  does  not  produce  death,  but  only 
paralyses  which  develop  after  a  long  incubation  of  fourteen  days  or 


more.1 

Arrhenius  and  Madsen  have  doubted  particularly  the  existence  of 

1  It  may  be  remarked  in  passing  that  such  additional  or  "by-poisons"  with 
a  long  period  of  incubation  are  not  limited  to  diphtheria  bacilli.  According 
to  the  observations  of  Sclavo  on  animals  infected  with  anthrax  it  is  highly 
probable  that  anthrax  bacilli  also  produce  poisons  having  a  toxin-like  action. 


504  COLLECTED  STUDIES  IN   IMMUNITY 

the  toxons.  According  to  them  the  long-drawn-out  toxon  zones  are 
the  expression  of  the  incomplete  combination  of  toxin  and  antitoxin, 
the  neutralization  of  which  they  believe  follows  the  ammonia-boric 
acid  type.  There  are,  however,  a  number  of  weighty  reasons  why 
this  view  cannot  be  accepted. 

It  was  but  natural  at  first  to  ascribe  the  toxon  stage  to  phenomena 
such  as  Arrhenius  and  Madsen  now  have  in  view.  It  had  already 
been  noticed  by  others  that  often  a  considerable  interval  exists  be- 
tween Lf  and  L0.  Knorr,  in  referring  to  this,  had  spoken  of  "un- 
neutralized  poison  residue/'  The  assumption,  however,  that  we  are 
here  dealing  with  the  result  of  an  incomplete  neutralization  is  con- 
troverted by  the  analysis  of  a  poison  which  I  encountered  during  the 
course  of  my  investigations.  This  was  Poison  No.  10  (of  my  series), 
whose  L0  and  Lf  values  were  very  close  together.  L0  contained  27.5 
and  Lf  29.2  L.  D.  Hence  D  =  1.7  L.  D.,  which  is  a  close  approach  to 
the  figure  demanded  by  a  simple  diphtheria  poison. 

The  following  considerations  will  show  that  this  value,  1.7  should  be  cor- 
rected so  as  to  be  still  lower.  The  original  calculations  were  based  on  my  earlier 
assumption  that  toxins  and  toxoids  are  uniformly  mixed.  This  however,  has 
been  superseded  by  the  spectrum  method  of  representing  the  neutralization  of 
poisons.  Experience  has  taught  us  that  such  deteriorated  poisons  usually 
consist  of  a  small  zone  of  hemitoxin  and  a  more  or  less  pronounced  zone  of 
tritotoxin-toxoid,  in  which  as  a  rule  nine  toxoid  equivalents  fall  on  one  toxin 
equivalent.  Several  times  I  have  observed  tritotoxin-toxoid  zones  containing 
Vio  toxin,  and  Madsen  also  has  described  such  a  poison.  As  can  be  seen  from 
our  calculations  given  above,  the  theoretical  change  from  L  „  to  Lf  is  influenced 
solely  by  the  tritotoxoid  zone.  If  we  therefore  assume  that  our  poison  pos- 
sessed a  tritototoxin-toxoid  portion  whose  strength  was  l/n,  (and  this  is  extremely 
probable)  we  shall  find  that  by  a  little  calculation  that  the  poison  probably 
contained  no  toxon  whatever.  Very  likely  the  tritotoxoid  zone  reached  to 
the  end  (200)  of  the  spectrum.  On  the  assumption  of  a  V,0  tritotoxin-toxoid, 
if  we  multiply  L0  by  21%oo  we  shall  obtain  Lt  =  28.9  L.  D.  This  agrees  very 
well  with  the  figures  obtained  experimentally,  Lf=29.2  L.  D. 

We  may  therefore  very  well  assume  that  we  were  dealing  with  a 
poison  free  from  toxon  or  one  which  contained  only  very  small  traces 
of  toxon. 

This  fact  is  hard  to  reconcile  with  the  theory  of  Arrhenius  and 
Madsen,  for   if   toxin    and    antitoxin    neutralized    each    other   like 
ammonia  and  boric  acid,  all  poisons  should  show  a  long  zone  of  in 
complete  neutralization. 

The  independent  existence  of  the  toxons  is  further  corroborated 
by  the  fact  that  the  toxon  zone  varies  enormously  in  different  speci- 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN  505 

.  mens  of  poison.  In  one  it  may  amount  to  about  one-fifth  of  the  toxin 
portion,  in  another  I  have  seen  equal  parts  of  toxon  and  toxin.  Dreyer 
and  Madsen  in  fact  have  recently  described  a  poison  which  contained 
three  times  as  much  toxon  as  toxin.  According  to  our  present  ex- 
periences, therefore,  the  amount  of  toxon  calculated  on  the  toxin  can 
vary  from  0  per  cent  to  300  per  cent.  Hence  I  find  it  impossible  to 
assume  that  we  are  dealing  with  neutralization  phenomena  such  as 
are  observed  with  ammonia  and  boric  acid,  for  such  neutralizations 
would  show  at  least  some  agreement. 

This  still  left  undecided  whether  the  toxon  is  a  primary  bacillary 
secretion  or  a  secondary  modification  of  the  toxin.  A  study  of  the 
development  of  one  poison  finally  gave  me  the  clue  to  this.  This  was 
poison  V,  whose  constitution  has  been  described  in  the  Deutsche 
med.  Wochenschrift  1898.  It  will  be  recalled  that  this  poison  pos- 
sessed the  following  limits  in  the  second  phase: 

L0  =  0.125;  Lt  =  0.26;  L.  D.  =  0.003. 

During  the  course  of  three  weeks  Geheimrath  Donitz  made  con- 
tinuous determinations  of  L0  and  L1",  using  very  uniform  animal 
material.  The  protocol  of  this  experiment  is  reproduced  in  full 
because  the  precision  of  the  methods  will  thereby  also  be  exhibited 
(see  table  on  page  506) . 

From  the  table  we  see  that  in  the  course  of  three  weeks  L0  has 
increased  from  0.15  to  0.20.  After  this  an  insignificant  increase 
brought  this  to  0.21;  from  then  on  L0  remained  constant.  During 
this  time  the  LA  dose  (0.26)  had  suffered  no  change  whatever,  for  on 
the  16th  of  July  a  mixture  of  0.25  poison +  1  I.  E.  killed  in  six  days 
and  0.275 -f  1  I.  E.  in  three  days.  Lt,  which  according  to  our  defi- 
nition is  the  mixture  that  will  just  kill  on  the  fifth  day,  must  have 
been  about  midway  between  these  two  values,  a  little  over  0.26. 
This  agrees  very  well  with  the  value  obtained  in  the  beginning.  To 
repeat,  during  the  course  of  this  stage  Lt  has  remained  constant, 
but  L0  has  increased  considerably  (from  0.125  to  0.21). 

This  fact  is  easily  explained.  The  toxin  portion  has  remained 
absolutely  unchanged  in"  its  end  zone,  as  can  at  once  be  seen  from 
the  constancy  of  the  Lt  dose.  On  the  other  hand  in  the  toxon  por- 
tion, which  is  expressed  by  the  difference  between  Lt  and  L0,  80 
toxon  equivalents  out  of  100  have  apparently  disappeared.  This 
eliminates  the  possibility  of  a  transformation  of  toxin  into  toxon, 
for  if  that  assumption  were  correct  one  would  expect  that  on  allow- 


506 


COLLECTED  STUDIES  IN  IMMUNITY. 


ing  the  bouillon  to  stand,  the  toxin  zone  would  decrease  and  the 
toxon  zone  become  considerably  greater.  In  this  case,  however,  we 
see  that  the  toxin  zone  remains  constant  while  the  toxon  zone  is 
reduced  to  one-fifth.1 

DETERMINATION  OF  L0  DOSE. 


Guinea-pigs  are  Injected  with  1  I.  E.  +  Varying  Amounts  of  Poison, 

Amount 
of  Poison 
cc. 

June. 

July 

21 

25 

29 

1 

4 

6 

10 

0.125 



0 











0.1275 

faint  trace 

almost  0 

— 

— 

— 

— 

— 

0.13 



— 

— 

— 



— 



0.14 

— 

— 

slight  but 
distinct 

— 

— 

— 

— 

0.15 

— 

:  

— 

just 

— 

— 

— 

neutral 

0.16 

— 



— 

slight  but 
distinct 

— 

— 

— 

0.17 

— 



— 

— 

little 

slight 

— 

0.18 

— 



— 

— 

1  ' 

1  t 

— 

0.19 

— 



— 

— 

more 

slight 

— 

oedema 

0.2 

— 



— 

— 

i  — 

more 

almost 

oedema 

neutral 

0.215 





— 

— 

— 

more 

some 

oedema 

oedema 

0.23 

— 



— 

— 

— 

— 

marked 

oedema 

"Faint  trace,"  "slight,"  etc.,  denote  the  degree  of  infiltration. 

It  is  difficult  to  say  a  priori  what  has  become  of  the  toxon  which 
has  disappeared.  On  account  of  certain  facts  which  I  shall  mention 
later,  1  have  assumed  that  we  are  here  dealing  with  the  formation 
of  an  analogue  of  toxoid,  namely,  a  substance  which  I  term  "toxo- 
noid."  I  conceive  this  to  be  a  toxon  in  which  the  toxophore  group 
has  become  modified. 


1  The  entire  course  of  the  decomposition,  in  which  from  day  to  day  we  could 
observe  the  toxon  becoming  weaker  and  weaker  speaks  against  the  possibility 
(in  itself  very  remote)  that  the  varying  composition  of  the  bouillon  is  respon- 
sible for  the  variation  in  the  number  of  toxons  in  the  individual  poisons.  In 
the  poison  here  described  the  decomposition  has  taken  place  in  the  same  bouillon 
and  in  so  short  a  time  that  very  great  alterations  in  the  bouillon  appear  to  be 
excluded. 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  507 

Another  fundamental  difference,  one  which  in  my  opinion  argues 
in  favor  of  the  individuality  of  toxin  and  toxon,  consists  in  the  differ- 
ent action  of  the  two  constituents.  The  action  of  diphtheria  toxin, 
as  is  well  known,  is  such  that  the  animals  die  with  symptoms  of 
hydrothorax,  ascites,  congestion  of  the suprarenals,  necrosis  of  the  skin. 
Somewhat  smaller  doses  kill  guinea-pigs  in  from  six  to  seven  days, 
the  animals  showing  ulceration  and  extensive  necrosis.  Still  smaller 
doses,  i,  J,  J,  J  L.  D.,  no  longer  produce  death,  but  regularly  cause 
necroses  which  are  surrounded  by  an  extensive  area  of  total  loss  of 
hair.  Small  fractions  of  the  fatal  dose  always  produce  emaciation 
of  the  animals.  In  contrast  to  this,  the  toxon,  i.e.  a  serum-poison 
mixture  in  which  only  the  toxin  fraction  is  completely  neutralized, 
never  kills  animals  acutely,  even  in  high  doses.  The  inflammatory 
properties  may  be  entirely  absent  in  small  doses,  while  in  large  doses 
they  are  present  to  only  a  slight  degree.  The  oedema  disappears 
completely  in  the  course  of  a  few  days,  there  .are  no  necroses,  and 
the  loss  of  hair,  if  it  occurs  at  all,  is  only  partial.  On  the  other  hand 
the  paralyses  are  very  characteristic,  and  these  appear  at  any  time 
between  the  fourteenth  and  twentieth  day,  depending  upon  the  dose, 
usually  in  the  third  week.  Frequently  the  animals  do  not  show  even 
a  trace  of  local  reaction  and  maintain  their  weight ;  then  suddenly  they 
are  attacked  with  the  paralyses  and  may  die  from  these  within  a  few 
days.  I  have,  never  seen  such  a  result  in  animals  inoculated  with  a 
pure  diphtheria  poison.  Now  and  then  a  guinea-pig  was  observed 
which  showed  these  paralytic  phenomena.  It  was  usually  one  that 
had  received  a  considerable  fraction  of  the  L.  D.  Invariably  it 
showed  extensive  necroses,  was  generally  very  sick  from  the  beginning, 
and  had  suffered  considerable  loss  of  weight.  In  view  of  the  slight 
amount  of  toxon  which  I  found  in  these  poisons,  such  animals  were 
evidently  supersensitive  to  the  toxon. 

Dreyer  and  Madsen  have  succeeded  in  differentiating  toxin  and 
toxon  qualitatively,  as  follows:  They  found  that  mixtures  of  a  diph- 
theria poison  and  antitoxin  in  which  the  limit  of  complete  toxin 
neutralization  was  nearly  approached,  exerted  only  toxon  effects 
when  given  in  small  doses.  If,  however,  the  mixture  was  increased 
tenfold,  death  was  brought  about  by  the  toxin.  This  is  readily 
explained.  The  determination  of  toxon  by  means  of  1  I.  E.  natu- 
rally cannot  be  absolutely  exact,  for  a  small  residue  of  toxin,  e.g. 
Vio  L.  D.,  can  readily  escape  observation.  If,  however,  a  sufficiently 
large  multiple  of  this  mixture,  e.g.  ten  times  the  original  quantity,  is 


508  COLLECTED  STUDIES  IX  IMMUNITY. 

injected,  this  will  now  contain  10/io  L.  D.  unneutralized.  If  now  the 
amount  of  antitoxin  was  also  somewhat  increased,  Dreyer  and  Mad- 
sen  found  that  even  with  this  multiple  amount  only  toxon  effects 
were  observed,  the  toxin  now  being  completely  neutralized  and  only 
toxon  remaining  free. 

Dreyer  and  Madsen  1  thereupon  subjected  this  same  poison  to  a 
thorough  study,  using  rabbits  for  the  purpose.  They  found  if  0.6  cc. 
poison  was  mixed  with  1  I.  E.,  that  this  mixture,  which  represents 
the  L0  dose  for  guinea-pigs,  is  still  highly  toxic  for  rabbits.  In  order 
to  render  this  dose  of  poison  completely  innocuous  for  rabbits  it  is 

240 
necessary  to  add  more  antitoxin,  in  this  case  -^—r  I.  E.     The  state- 


ments  concerning  the  behavior  of  mixtures  between  these  two  limits 
are  also  of  considerable  importance.      A  mixture  of  0.6  cc.  poison  + 

210 

-—>  I.  E.  injected  into  a  rabbit  causes  death  on  the  twenty-second 

200 

•day   with   paralytic   symptoms.     The   incubation   period   is   sixteen 

232 
days.     Even  a  mixture  of     —  I.  E.  with  the  same  amount  of  poison 


caused  paralyses,  which  appeared  on  the  sixteenth  day  and  con- 
tinued for  several  weeks.  This  behavior  is  so  important  for  our  view 
concerning  the  existence  of  different  poisons  that  I  must  enter  a 
little  more  fully  into  the  subject.  According  to  our  definition  of  the 

232 
L0  dose,  mixtures  like  the  one  containing     -—  I.  E.,  and  therefore 


possessing  a  considerable  excess  of  antitoxin,  are  absolutely  innocuous 
for  guinea-pigs  and  can  be  injected  in  any  quantity.  In  virtue  of 
the  excess  of  antitoxin  such  mixtures  suffice  to  passively  immunize 
the  animal  and  to  protect  it,  provided  suitable  doses  have  been  in- 
jected, against  diphtheria  poison  and  diphtheria  bacilli.  If  then 
such  mixtures  are  still  toxic  for  rabbits  only  one  possibility  remains, 
namely,  that  the  diphtheria  poison  in  question  contains  a  substance 
which  is  non-toxic  for  guinea-pigs  but  toxic  for  rabbits.  This  sub- 
stance I  term  toxonoid.2 

1  See  also  my  article  in  Munch,  med.  Wochensch.  1903,  Nos.  33,  34. 

2  At  the  outset  of  my  investigations  I  made  entirely  similar  observations. 
My  very  extensive  but  unpublished  studies  made  at  that  time  convinced  me 
that  this  property  is  not  common  to  all  diphtheria  poisons,  for  I  also  found 
some  in  which  the  L0  dose  was  exactly  the  same  in  rabbits  and  in  guinea-pigs. 
This  fact  furthermore  refutes  the  assumption  that  the  phenomenon  described 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN.  509 

So  far  as  the  behavior  of  partially  neutralized  mixtures  is  con- 
cerned, the  observations  of  these  authors  show  that  mixtures  which 
exert  only  toxon  effects  on  guinea-pigs  produce  death  in  rabbits 
with  symptoms  of  diphtheria  poisoning.  I  believe  that  all  these 
phenomena  are  best  explained  by  the  assumption  that  there  are  at 
least  three  different  varieties  of  poisons,  and  that  these  possess  differ- 
ent affinities  and  different  actions.  These  poisons  are: 

1.  Toxin,  possessing  the  highest  affinity,  kills  rabbits  and  guinea- 
pigs  acutely,  but  is  more  toxic  for  the  former. 

2.  Toxon,  killing  rabbits  acutely  and  guinea-pigs  with  symptoms 
of  paralysis. 

3.  Toxonoid,  producing  paralyses  in  rabbits,  non-toxic  for  guinea- 
pigs. 

The  fact  that  all  three  poisons  act  more  strongly  on  rabbits  than 
on  guinea-pigs  is  explained  by  the  absolute  higher  susceptibility  of 
the  former. 

Dreyer  and  Madsen  have  recently  described  a  diphtheria  poison 
in  which  toxoid  effects  could  be  demonstrated  even  on  the  injection 
of  sublethal  doses  of  the  pure  poison.  This  behavior  is  at  once  under- 
stood if  we  study  the  constants  of  this  poison  as  they  were  determined 
by  these  authors,  for  whereas  in  the  other  poisons  examined  there 
were  33  toxon  equivalents  to  167  toxin  equivalents  (toxon :  toxin  = 
1:5),  in  this  poison  the  proportion  was  just  the  reverse,  there  being 
three  times  as  much  toxon  as  toxin.  Xo  wonder  therefore  that  with 
the  toxon  fifteen  times  more  concentrated  even  sublethal  doses  of 
the  pure  poison  should  suffice  to  make  toxon  effects  evident. 

In  view  of  the  high  theoretical  significance  which  attaches  to  the 
poison  described  by  Dreyer  and  Madsen,  I  cannot  refrain  from  giving 
briefly  my  conception  of  its  constitution.  The  authors  have  repre- 
sented the  poison  in  the  form  of  a  curve,  one  which  at  first  sight  seemed 
rather  strange  to  me.  As  soon,  however,  as  I  transformed  their 
graphic  representation  into  a  spectrum  by  the  aid  of  their  figures, 
the  constitution  of  the  poison  was  found  to  agree  very  well  with 
other  well-known  diphtheria  poisons.  The  only  difference  is  the  very 


is  due  to  an  incomplete  neutralization,  such  as  Arrhenius  and  Madsen,  for  exam- 
ple, have  demonstrated  in  the  case  of  boric  acid  and  ammonia,  and  in  the  union 
of  tetanolysin  with  its  antitoxin.  If  that  were  the  case  one  would  expect  to 
see  the  phenomenon  in  all  diphtheria  poisons  in  equal  degree,  and  this  is  not 
the  case. 


510  COLLECTED  STUDIES  IN   IMMUNITY. 

large  content  of  toxon.  The  spectrum,  which  corresponds  to  the 
curve  obtained  by  the  authors,  is  here  reproduced  (Fig.  3,  Phase  II). 

From  this  we  see  that  a  zone  of  hemitoxin  in  the  beginning  of  the 
spectrum  is  followed  by  a  zone  of  almost  pure  toxin,  and  this  in  turn 
by  a  zone  of  tritotoxin-toxoid.  Then  comes  the  very  long  toxin 
fraction. 

To  one  employing  this  mode  of  representation,  such  a  spectrum 
not  only  pictures  the  present  constitution  of  the  poison  but  also 
frequently  permits  him  to  reconstruct  its  previous  constitution. 
In  this  case,  for  example,  it  was  possible  to  do  so  with  the  aid  of 
several  statements  by  the  authors  concerning  earlier  and  later  stages. 
According  to  these  figures  I  would  assume  that  in  the  first  phase 
the  poison  contained  a  pure  toxin  in  the  initial  zone.  In  the  second 
phase,  the  period  at  which  the  poison  was  studied  by  Dreyer  and 
Madsen,  this  had  become  transformed  into  hemitoxin.  In  the  third 
phase  it  may  become  pure  prototoxoid.  A  fourth  phase  would  then 
show  the  transformation  of  the  pure  toxin  in  the  above  spectra  into 
hemitoxin  and  the  poison  would  then  have  reached  the  point  which 
we  have  so  frequently  observed  in  other  poisons.  The  spectra  of 
these  various  phases  is  as  follows  (Fig.  7) : 

I  shall  now  present  the  figures  which  Madsen  and  Dreyer  ob- 
tained when  they  started  with  double  the  L0  dose  (0.1  cc.  poison). 
In  the  first  phase,  their  statement  that  the  lethal  dose  was  0.0015  cc. 
shows  that  0.1  cc.  poison  contains  66  L.  D.  Calculation  from  the 
spectrum  gives  65  L.  D. 

The  second  phase,  of  course,  agrees  entirely  with  the  statements 
of  the  authors,  since  the  spectrum  was  constructed  according  to  these. 

In  the  third  phase  the  formation  of  the  prototoxoid  zone  from 
the  previous  zone  of  hemitoxin  is  readily  seen  from  a  second  neu- 
tralization test,  one  made  with  normal  horse  antitoxin. 

In  phase  IV  the  lethal  dose  had  risen  to  0.0027,  corresponding 
to  37  L.  D.  in  0.1  cc.  Calculating  this  from  my  spectrum  I  obtain 
35  L.  D.,  which  is  but  2  L.  D.  smaller  than  would  correspond  to 
the  final  stage.  Perhaps  this  stage  had  been  nearly  but  not  yet 
completely  attained.  It  is  probable  that  if  the  examination  had 
been  made  a  little  later  the  figure  would  have  been  exactly  35. 

The  figures  obtained  from  my  reconstructed  spectra  harmonize 
so  well  with  those  obtained  experimentally  by  the  authors  that  it 
seems  almost  impossible  to  doubt  the  correctness  of  my  assumptions 
concerning  the  constitution  of  the  poison  and  the  process  of  its  trans- 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN. 


511 


formation.  This  proves  that  in  this  poison  the  toxin  zone  behaved 
exactly  the  same  in  its  transformation  as  it  did  in  the  other  diph- 
theria poisons  examined. 

I  believe  it  will  be  seen  from  my  explanations  that  my  mode  of 
procedure  in  the  study  of  diphtheria  poison  has  been  exceedingly 

Phase! 


10 


0   10  20  30  40  50  60  70  80  90  100  110  120  130  140  150  160  170  180  190  200 

Phase  n 


10 


10     20     30     40     50     60      70     80     90    100   110   120   130   140   150   160    170  180   190  200 

Phase  IH 


0   10  20  30  40   50  60  70  80  90  100  110  120  130  140  150  160  170  180  190  200 

Phase  IV 


1U  20  30  40  50  60  70  80  90  100  110  120  130  140  150  160  170  |18J)  190  200 

FIG.  7. 

careful,  and  that  the  objections  raised  against  my  results  do  not  apply. 
I  must  therefore  continue  to  maintain  my  original  standpoint,  and 
deem  it  well  therefore  to  once  more  define  my  views  concerning  the 
poison  of  diphtheria. 


512  COLLECTED  STUDIES  IN   IMMUNITY. 

1.  The   diphtheria   bacillus   produces   several   kinds   of  poisons, 
especially  toxins  and  toxons. 

2.  The  affinity  of  diphtheria  toxin  to  the  antitoxin  is  very  great. 

3.  The  deviations  from  a  straight  line  as  they  manifest  themselves 
in   the  graphic  representation   of  the  neutralization   of   the  poison 
cannot  be  explained  by  the  assumption  of  a  single  poison  possessing 
a  weak  affinity.     They  are  rather  the  expression  of  the  fact  that  the 
poison  bouillon  contains  admixtures  of  various  kinds  of  substances 
of  a  toxoid  character. 

4.  The  varied  affinity  of  the  toxoids  cannot  be  explained  by  the 
assumption  that  a  simple  toxin  when  transformed  into  toxoid  suffers 
a  change  in   affinity  either  positively  or  negatively.     Rather  does 
this  indicate  that  the  toxic  bouillon   contains,   preformed,   various 
toxins  of  different  affinities. 

5.  There  is  no  change  in  the  haptophore  group  in  the  formation 
of  toxoid. 

6.  The   absolute   number  of   combining   units   contained    in    the 
immune  unit  or  in  the  L0  dose  of  poison  is  200. 1 

I  have  finished.  If  the  results  of  the  first  encounter  of  two  such 
different  methods  of  study  as  the  mathematico-physical  and  the  bio- 
logical have  not  shown  complete  agreement  we  should  not  be  at  all 
.surprised.  The  natural  aim  of  physical  chemistry  must  always  be 

1  Bordet  has  recently  attempted  to  explain  the  toxon  phenomena  by  the 
assumption  that  the  toxin  molecule  can  combine  with  antitoxin  in  varying 
proportions.  One  would  accordingly  have  to  assume  that  the  toxin  molecule 
contains  several  haptophore  groups.  The  complete  occupation  of  these  groups 
causes  the  toxicity  to  be  entirely  lost,  whereas  partial  saturation  causes  a  de- 
crease in  toxicity.  That  is  to  say,  amounts  of  antitoxin  which  do  not  com- 
pletely neutralize  the  toxin  would  weaken  it  in  such  fashion  that  it  would  exert 
a  different  action.  It  is  strange  that  so  eminent  an  investigator  as  Bordet 
should  not  have  attempted  to  convince  himself  of  the  correctness  of  this  hy- 
pothesis by  means  of  the  experiment.  He  would  then  have  found  that  the 
facts  are  irreconcilable  with  such  an  assumption.  We  have  shown  at  great 
length  that  the  toxon  actions  are  nothing  less  than  constant  phenomena  and 
have  called  attention  to  the  great  extent  of  the  quantitative  variations  (0-300). 
If  one  were  to  follow  Bordet  it  would  then  be  necessary  to  assume  an  enormous 
multiplicity  of  haptophore  groups  in  the  toxin  molecules,  and  this  would  lead 
to  a  hypothesis  far  more  complicated  than  mine,  although  the  latter  harmo- 
nizes all  the  experimental  results.  In  support  of  his  conception  Bordet  refers 
to  experiments  with  complement  and  anticomplement.  I  must  say,  however, 
that  in  these  we  are  dealing  with  such  complicated  relations  that  it  is  not  per- 
missible to  apply  the  conclusions  drawn  from  them  to  the  far  simpler  relations 
existing  between  toxin  and  antitoxin. 


THE  CONSTITUENTS  OF  DIPHTHERIA  TOXIN  513 

to  introduce  as  few  factors  as  possible  for  purposes  of  calculation, 
whereas  biological  analysis  always  seeks  to  pay  due  regard  to  the 
wonderful  multiplicity  of  organic  matter.  However,  I  believe  that 
these  two  methods  can  readily  be  combined  and  that  this  will  be 
very  desirable.  The  biologist  will  have  to  content  himself  in  so  far 
yielding  to  the  economy  of  the  mathematical  view  that  he  restricts 
his  assumptions  to  the  smallest  possible  number.  The  physical 
chemist,  on  the  other  hand,  cannot  escape  the  obligation  of  paying 
due  heed  to  this  minimal  multiplicity,  the  result  of  experimental 
research.  Naturally  the  problem  is  thus  made  extremely  difficult,  so 
that  success  will  require  that  the  greatest  authorities  in  physical 
chemistry  work  hand  in  hand  with  the  best  biological  talent.  For 
this  reason  I  regard  it  as  a  great  gain  to  science  that  so  eminent  a 
leader  as  Svante  Arrhenius  is  taking  a  lively  interest  in  our  work, 
and  has  joined  hands  with  my  friend  and  pupil,  Thorvald  Madsen. 


XXXVIII.  TOXIN  AND  ANTITOXINS 

A  REPLY  TO  THE  LATEST  ATTACK  OF  GRUBER. 
By  PAUL  EHRLICH. 

IN  a  domain  that  is  open  to  experimental  investigation  it  is 
neither  easy  nor  without  danger  for  one  to  express  criticism  merely 
as  a  result  of  literary  studies. 

This  is  especially  true  in  that  most  difficult  field  in  the  entire 
study  of  immunity,  namely,  the  subject  of  toxins.  Only  one  who 
has  devoted  years  of  unprejudiced  study  at  the  laboratory  table 
to  this  subject  and  gathered  a.  host  of  observations  and  experiences 
will  be  in  a  position  to  orientate  himself  in  the  confused  mass  of 
true  and  false  statements  contained  in  the  literature.  The  outsider 
will  find  it  very  difficult  to  correctly  analyze  all  this  material. 
Hence  it  is  all  the  more  remarkable  that  Gruber2  should  choose 
the  subject  of  toxins  for  the  main  portion  of  his  attack  upon  me, 
for  according  to  his  own  admissions  that  is  the  field  which  he  knows 
merely  from  literary  studies.  Against  such  critics  I  am  in  the  unpleas- 
ant position  of  a  man  who  is  compelled  to  discuss  colors  with  the 
blind.  Nevertheless  I  cannot  well  escape  the  thankless  task  of 
replying,  at  least  to  the  main  points  in  Gruber's  polemic,  for  it  is 
indisputable  that  this  attack,  addressed  chiefly  to  those  without 
special  training  in  this  field,  is  capable  of  causing  wide-spread  con- 
fusion, owing  to  its  positive  tone  and  its  severity. 

Gruber's  first  important  error  lies  in  the  assumption  that  a  con- 
tro version  of  the  plurality  of  poisons,  to  which  I  hold,  signifies  the 
downfall  of  the  side-chain  theory  without  further  ado.  The  side- 
chain  theory,  however,  proceeds  from  the  assumption  that  the  toxin- 

1  Reprinted  from  the  Munch,  med.   Wochensch.   1903,  Nos.  33  and  34. 

2  M.  Gruber  and  Cl.  v.  Pirquet,  Toxin  und  Antitoxin,  Munch,  med.  Wochensch. 
1903,  Nos.  28  and  29. 

514 


TOXIN   AND   ANTITOXIN.  515 

like  poisons  are  characterized  by  a  haptophore  and  a  toxophore 
group,  of  which  only  the  former  effects  the  anchoring  of  the  toxin. 
Practically  therefore  only  this  group  is  important  for  the  produc- 
tion of  antitoxins.  This  view  is  only  the  logical  consequence  of 
the  fact  that  on  long  standing  the  poison  bouillon  undergoes  modi- 
fications, resulting  in  the  production  of  what  1  term  toxoids.  These 
substances  are  characterized  by  this,  that  the  haptophore  group 
has  remained  intact,  while  the  toxophore  group,  depending  on  cir- 
cumstances, has  suffered  .partial  or  complete  modification.  Not 
infrequently  it  can  be  shown  that  the  formation  of  toxoid  is  quan- 
titative, the  combining  power  of  the  toxic  bouillon  being  unchanged 
despite  a  considerable  loss  of  toxicity. 

Gruber,  by  means  of  certain  calculations,  appears  to  question 
this  fact;  he  refers  exclusively  to  my  very  earliest  publications  in 
which,  naturally,  the  evidence  was  still  incomplete.  It  would  have 
been  better  if  Gruber  had  studied  instead  my  later  publications, 
for  then  he  could  easily  have  convinced  himself  that  my  statement 
is  entirely  correct.  I  shall  mention  but  one  of  my  poisons  1  as  an 
example.  In  this  the  L  dose  was  originally  0.25  cc.,  the  lethal  dose 
0.0025  cc.  At  the  end  of  the  investigation  Lf  had  increased  to 
0.26  cc.,  the  lethal  dose,  however,  to  0.004  cc.  The  number  of  lethal 
doses,  therefore,  in  approximately  the  same  amount  of  L-j-  had  been 
reduced  from  100  to  65.  Madsen2  describes  a  poison  in  which  the 
neutralizing  power  remained  constant  during  the  course  of  two 
years,  while  the  toxicity  was  reduced  one-half,  from  0.02  to  0.04. 
Furthermore  Arrhenius  and  Madsen  in  their  most  recent  work 3 
describe  the  toxoid  modification  of  a  tetanus  toxin.  These  consist 
in  the  fact  that  the  combining  power  remains  intact  while  the  toxicity 
is  decreased  to  one-sixth.  It  is  seen  therefore  that  the  doubt  thrown 
upon  my  quantitative  statements  is  due  entirely  to  a  disregard  of 
readily  accessible  facts.  This  quantitative  transformation  consti- 
tutes a  somewhat  annoying  fact  for  Gruber,  and  he  therefore  seeks 
to  explain  it  as  follows: 

"  Imagine,  if  you  will,  that  9/io  of  the  toxin  molecules  present 
are  changed  into  toxoids,  the  minimal  lethal  dose  will  then  be  increased 

1  Described  in  Deutsche  med.  Wochensch.  1898,  No.  38. 

2Annales  de  ITnstitut  Pasteur.,  T.  13,  1899. 

5  S.  Arrhenius  and  Th.  Madsen,  Physical  Chemistry  applied  to  Toxins  and 
Antitoxins,  Festskrift  ved.  indvielsen  af  Statens  Serum  Institut,  Kopenhagen, 
1902;  German  in  Zeitsch.  fur  physiol.  Chem.  1903. 


516  COLLECTED  STUDIES    IN  IMMUNITY. 

tenfold  whereas  the  L0  value  will  remain  unchanged;  this  is  Ehr- 
lich's  hypothesis.  If  9Ao  the  toxin  molecules  had  lost  their  toxicity, 
without  there  being  any  formation  of  toxoids  capable  of  combining 
with  antitoxin,  the  L0  value  would  be  increased  ten  times.  If,  how- 
ever, simultaneously  with  the  loss  of  9/i0  the  toxicity,  the  fluid 
were  to  lose  9/i0  the  reaction  rapidity  for  antitoxin,  so  that  the 
constant  of  the  reaction  would  be  decreased  9/1(),  it  would  be  found 
that  the  L0  value  would  manifest  itself  unchanged." 

Gruber  would  have  done  better  to  have  made  some  of  these  com- 
paratively simple  experiments  himself  than  to  advance  such  an 
untenable  assumption.  We  are  here  dealing  with  experiments 
which  constitute,  in  fact,  the  very  beginning  of  the  technique  of 
testing  poisons.  Thus,  when  in  1897 1  I  formulated  the  law  that 
the  combination  of  poison  and  antibody  takes  place  more  rapidly 
in  concentrated  solutions  than  in  weak  solutions,  it  was  as  the  result 
of  just  such  studies  made  on  diphtheria  and  tetanus  toxin.  In  these 
studies  I  convinced  myself  that  the  affinity  between  diphtheria  anti- 
toxin and  diphtheria  toxin  is  far  greater  than  that  between  tetanus 
antitoxin  and  tetanus  toxin.  The  union  of  diphtheria  toxin  and 
its  antitoxin  is  effected  very  quickly,  so  that  at  the  end  of  five  to 
ten  minutes  one  may  be  sure  that  complete  union  has  taken  place. 
It  is  entirely  immaterial  whether  one  is  dealing  with  fresh  poisons 
or  with  poisons  poor  or  rich  in  toxoids.  1  shall  here  reproduce  an 
experiment  which  I  have  recently  made  because  Danysz 2  insisted 
that  the  neutralizing  power  of  the  diphtheria  poison  changes  when 
the  poison  is  allowed  to  stand  for  some  time. 

The  experiment  was  performed  with  the  standard  serum  and 
standard  toxin  used  in  the  official  standardization.  Both  substances 
had  therefore  been  very  accurately  titrated.  The  mixture  was 
allowed  to  stand  fifteen  minutes  and  twenty-four  hours  and  the 
result  showed  that  in  this  time  not  the  least  change  had  taken  place 
in  the  constant.  In  the  experiments  of  Danysz,  therefore,  some 
error  has  probably  crept  in.  In  any  event  there  is  no  change  in 
the  reaction  time  on  the  decrease  of  toxicity  of  the  diphtheria  toxin. 

Guinea-pig  I  receives  1  I.  E.  serum +  0.78  cc.  poison  (Lt)  fifteen 
minutes  after  mixing.  It  dies  on  the  fourth  day. 

Guinea-pig  II  receives  the  same  mixture  twenty-four  hours  after 
mixing.  It  dies  on  the  fourth  day. 

1  Die  Werthbemessung  des  Diphtherieheilserums,,  Jena,  1897, 

2  Annales  de  1'lnstitut  Pasteur  1902. 


TOXIN  AND  ANTITOXIN  517 

Guinea-pig  III  receives  0.8  cc.  poison,  otherwise  same  as  I.  It 
dies  in  three  and  one-half  days. 

Guinea-pig  IV  receives  0.8  cc.  poison,  otherwise  same  as  II.  It 
dies  in  three  and  one-half  days. 

Another  thing  which  is  entirely  irreconcilable  with  Gruber's 
assumption  is  the  fact  that  there  exist  prototoxoids,  i.e.,  toxoids 
which  possess  a  higher  affinity  for  the  antitoxin  than  the  toxin  itself 
does.  The  existence  of  these  was  first  pointed  out  by  me  and  has 
since  been  confirmed  by  Madsen  and  also  by  Arrhenius.  The  exist- 
ence of  the  prototoxoids  becomes  clearly  manifest  by  the  fact  that 
one  can  add  a  certain  quantity  of  antitoxin  to  the  toxin  solution 
without  affecting  the  toxicity  in  the  slightest  degree. 

Mention  must  also  be  made  of  the  fact  that  similar  phenomena 
have  been  observed  in  a  large  number  of  other  poisons.  It  will 
suffice  here  if  I  remind  the  reader  that  toxoid  changes  have  been 
observed  in  ricin  (Jacoby),  abrin  (Romer),  staphylotoxin  (Wechs- 
berg,  Neisser),  cobra  venom  (Meyers,  Flexner).  Furthermore  Mor- 
genroth  and  I  showed  that  in  complement  also  there  is  a  destruction 
of  the  real  active  portion,  the  zymotoxic  group,  while  the  hapto- 
phore  group  remains  intact.  The  existence  of  complementoids 
has  been  demonstrated  decisively  by  Sachs  and  myself,1  although 
Gruber  had  termed  them  "  merely  fervent  wishes  floating  about 
in  the  serum." 

Furthermore  it  will  be  remembered  that  similar  phenomena 
are  observed  in  the  agglutinins  and  coagulins  (precipitins),  the  hap- 
tophore  group  of  the  agglutinin  or  the  precipitin  remaining  intact, 
while  the  agglutinophore  group  is  destroyed.  This  phenomenon 
was  first  pointed  out  in  the  excellent  study  made  by  Eisenberg  and 
Vblk  in  Paltauf  s  laboratory.  Since  that  time  a  large  mass  of  liter- 
ature has  grown  up  around  this  subject  so  that  now  there  is  not 
the  least  doubt  concerning  the  existence  of  these  substances,  which 
normally  occur  in  the  form  of  proagglutinoids.  A  recent  study 
by  Korschun 2  makes  it  probable  that  something  similar  to  this 
occurs  in  ferments,  particularly  in  rennin.  In  all  these  various 
cases  it  seems  to  be  the  rule  that  the  real  functionating  group  is  far 
more  labile  than  the  one  which  effects  combination,  namely,  the 
haptophore  group.  Hence  I  believe  that  the  formation  of  such 

1  See  page  209. 

2  Zeitsch.  f.  physiol.  Chemie,  Bd.  37,  1903. 


518  COLLECTED  STUDIES  IN  IMMUNITY. 

modifications  must  be  classed  with  the  positively  demonstrated 
facts  in  medicine. 

It  is  entirely  incomprehensible  how  Gruber  could  believe  that 
the  possible  controversion  of  the  plurality  of  poisons  assumed  by 
me  denotes  the  downfall  of  the  entire  side-chain  theory.1 

How  false  such  a  conclusion  is  can  be  seen  from  the  fact  that 
when  I  devised  the  side-chain  theory  I  believed  the  diphtheria  poison 
to  be  a  simple  substance.  My  later  studies,  however,  convinced 
me  that  the  poison  consists  of  several  modifications:  prototoxin, 
deutero toxin,  tritotoxin,  and  toxon.  It  can  easily  be  seen  from 
my  publications,  however,  that  I  ascribe  the  same  combining  group 
to  all  of  these;  they  differ  merely  in  their  toxophore  groups.  In 
the  production  of  diphtheria  antitoxin  all  of  these  modifications 
act  in  exactly  the  same  way.  It  shows  a  deplorable  lack  of  com- 
prehension, therefore,  when  Gruber  says  that  the  refutation  of  the 
plurality  of  toxins  will  "  give  this  side-chain-theory  spook  its 
quietus." 

However,  let  us  see  what  proofs  Gruber  advances  against  the 
plurality  of  the  poisons.  On  a  previous  occasion  when  Gruber 
brought  forward  these  same  arguments  I  allowed  them  to  pass  with- 
out specially  controverting  them,  for  I  felt  that  his  faulty  mode 
of  reasoning  would  at  once  be  apparent  to  the  specialist.  Now  that 
Gruber,  however,  returns  to  this  subject  I  think  it  may  be  well  to 
discuss  the  facts  somewhat  in  detail. 

In  the  majority  of  poisons  it  is  probably  a  fact  that  the  toxicity 
depends  upon  the  animal  species,  a  certain  poison  being  more  toxic 
for  one  species  than  for  another.  In  chemically  definite  poisons, 
alkaloids,  etc.,  this  behavior  is  usually  a  constant  one,  so  that  in 
text-books  on  toxicology  the  fatal  doses  per  kilo  of  body  weight 

1  Arrhenius  and  Madsen  (1.  c.)  in  their  very  interesting  study  have  ques- 
tioned whether  the  phenomena  of  neutralization,  which  I  described  and  referred 
to  a  plurality  of  poisons,  are  due  to  a  difference  in  the  poisons  or  whether,  as 
they  think  probable,  they  are  merely  the  expression  of  a  neutralization  between 
two  substances  of  weak  affinities.  For  the  present  I  shall  merely  point  out 
that  my  own  statements  refer  only  to  diphtheria  toxin,  which  possesses  a  much 
higher  affinity  for  the  antitoxin  than  does  tetanus  toxin.  The  investigations 
of  these  esteemed  authors  have  disclosed  one  source  of  error  which  could  easily 
creep  into  neutralization  experiments.  Nevertheless  I  believe  that  their  con- 
ception does  not  apply  to  the  toxin  of  diphtheria  which  I  have  studied  so  closely. 
I  shall  go  into  these  questions  more  fully  elsewhere,  and  hope  then  to  show 
that  the  standpoint  maintained  by  me  is  entirely  correct. 


TOXIN   AND  ANTITOXIN.  519 

are  usually  given  for  various  animal  species.  In  the  beginning  it 
was  thought  that  the  same  conditions  held  true  for  the  bacterial 
poisons  and  several  such  scales  of  toxicity  were  given  out  by  high 
authorities.  As  soon,  however,  as  different  toxin  solutions  of  the 
same  species  were  examined,  e.g.  diphtheria  toxins  obtained  from 
different  cultures  or  in  different  laboratories,  it  was  found  that, 
unlike  the  alkaloids,  the  scale  of  toxicity  was  a  variable  one.  In 
the  case  of  one  poison,  for  example,  I  found  that  a  guinea-pig  of 
250  grammes  was  uniformly  killed  by  a  dose  of  0.00375-0.004  cc., 
and  a  rabbit  of  1800  grammes  by  a  dose  of  0.009  cc.  This  corre- 
sponds to  a  ratio  of  1:2:2-2.4.  In  another  poison  the  figures  were 
0.003  for  guinea-pigs  and  0.004  for  rabbits,  corresponding  to  a  pro- 
portion of  1:1.3.  This  showed  that  in  two  different  poisons  the 
susceptibility  of  rabbits  varied  more  than  half. 

The  conditions,  however,  are  far  more  interesting  and  instruc- 
tive in  the  case  of  tetanus  poison.  For  a  long  time  a  controversy 
existed  between  v.  Behring  and  Tizzoni.  The  former  stated  that 
tetanus  poisons  act  150  times  weaker  on  rabbits  than  on  mice,  whereas 
Tizzoni  declared  that  a  poison  prepared  by  him  was  just  as  toxic 
for  rabbits  as  for  mice.  From  the  papers  of  these  authors  it  is  cer- 
tain that  the  two  poisons  when  tested  on  mice  were  identical.  A 
definite  amount  of  either  poison — for  example,  a  single  fatal  dose 
for  mice — was  neutralized  by  the  same  quantity  of  antitoxin.  So 
far  as  mice  were  concerned,  therefore,  the  two  poisons  were  identical. 
As  soon  as  the  poisons  were  tested  on  rabbits,  however,  the  above- 
mentioned  enormous  difference  in  toxicity  becomes  apparent.  This 
at  once  shows  that  these  two  poisons  cannot  possibly  be  identical. 
Wherein,  then,  does  the  difference  consist?  We  have  seen  that  the 
two  poisons  are  neutralized  by  the  same  antitoxin,  and  that  fur- 
thermore immunization  with  one  of  the  poisons  is  followed  by  the 
production  of  an  antitoxin,  which  acts  also  on  the  other  poison. 
From  this  it  follows  that  the  haptophore  group  must  be  the  same 
in  both.  Hence  we  must  be  dealing  with  a  difference  in  the  toxo- 
phore  group,  v.  Berhing's  poison  possessing  a  toxophore  group  which 
is  highly  virulent  for  mice  and  only  slightly  so  for  rabbits,  whereas 
Tizzoni's  poison  contains  a  group  which  acts  equally  on  both  ani- 
mals. This  difference  would  be  very  like  that  which  I  have  demon- 
strated in  the  case  of  diphtheria  toxin  and  toxon.  One  might,  how- 
ever, think  of  an  entirely  different  explanation,  namely,  that  the 
strain  of  bacteria  with  which  Tizzoni  worked  secreted  an  entirely 


520  COLLECTED  STUDIES  IN  IMMUNITY. 

different  kind  of  poison  than  the  Marburg  culture.  But  this  proved 
not  to  be  the  case,  for  v.  Behring  demonstrated  that  his  tetanus  poison 
when  injected  into  rabbits  in  large  quantities  suffers  a  considerable 
diminution  in  toxicity.  On  testing  the  properties  of  the  poison 
contained  in  the  serum  of  the  poisoned  animals  he  found  that  this 
residual  poison  possessed  the  same  constants  as  Tizzoni's  poison. 
From  this  it  follows  that  v.  Behring's  poison  contained  also  a  cer- 
tain proportion  of  the  Tizzoni  variety.  The  Marburg  culture  must 
therefore  have  produced  two  varieties  of  poison  at  the  same  time. 
Naturally  by  mixing  the  two  poisons  one  can  obtain  new  poisons 
which,  while  they  manifest  the  same  action  on  mice,  will  have  any 
desired  relative  toxicity  for  rabbits;  this,  of  course,  within  certain 
limits.  If  one  were  to  take  the  time  and  trouble  to  examine  a  large 
number  of  native  poisons  from  different  laboratories,  corresponding 
differences  between  them  would  probably  be  encountered. 

If  we  recollect  that  various  specimens  of  the  chemically  simple 
poisons  manifest  the  same  relative  toxicity  on  different  animals, 
and  then  consider  the  behavior  of  tetanus  toxins  as  just  described, 
we  shall  conclude  that  bacterial  poisons  of  different  origin,  which 
manifest  a  variation  in  their  relative  toxicity,  are  not  of  simple  con- 
stitution, but  are  made  up  of  several  different  constituents.  It 
shows  very  little  knowledge  of  the  subject  therefore  when  Gruber 
says:  "  v.  Behring  shows  that  two. toxin  solutions,  which  in  a  given 
unit  of  volume  contain  equal  f  Ms.,  i.e.,  whose  unit  of  volume  kills 
a  like  number  of  grammes  of  mouse  in  four  days,  may  have  an  entirely 
different  content  of  f  rabbit,  t  pigeon,  t  goat,  and  f  horse.  This 
at  once  disposes  of  Ehrlich's  conclusions."  It  is  just  such  phenomena 
which  argue  in  favor  of  the  plurality  of  poisons;  they  do  not  speak 
against  it. 

Gruber  bases  another  of  his  objections  on  the  interesting  obser- 
vations made  by  Madsen  and  Dreyer  on  toxons  (Zeitsch.  f.  Hygiene, 
Vol.  37,  page  251).  In  his  dictatorial  manner  he  says  that  "  these 
observations  demonstrate  conclusively  that  Ehrlich's  method  of 
analyzing  toxins  is  absolutely  useless.  Only  a  person  ignorant  of 
chemistry  could  maintain  that  the  different  results  in  guinea-pigs 
and  in  rabbits  are  sufficiently  explained  by  the  different  susceptL 
bility  of  the  animals  to  the  toxins." 

To  begin,  Gruber's  premise  is  absolutely  misleading,  when  he 
says: 

"  But  if  the  poison  is  neutralized  it  will  be  without  effect  even 


TOXIN   AND  ANTITOXIN.  521 

on  the  most  susceptible  animals.  Let  us  imagine,  for  example,  a 
mixture  of  sulphuric  and  acetic  acids,  neutralized  by  the  gradual 
addition  of  baryta  water.  Once  all  the  sulphuric  acid  is  neutralized, 
even  the  most  sensitive  reagent  to  free  strong  mineral  acids  will  be 
unable  to  detect  any  trace  of  it." 

Let  us  see  just  what  Gruber  means  by  this  comparison.  The 
sulphuric  acid  corresponds  to  the  toxin ;  the  antitoxin  is  represented 
by  the  alkali.  In  accordance  with  the  comparison  the  receptors  of 
the  cells  are  represented  in  the  animal  body  by  the  alkali  of  the 
tissues.  If  now  we  inject  an  animal  with  sulphuric  acid  previously 
neutralized  with  ammonia,  i.e.,  a  solution  of  ammonium  sulphate, 
it  will  depend  mainly  on  the  affinity  of  the  tissue  alkali,  whether  or 
not  the  neutral  ammonium  sulphate  will  be  decomposed  and  sul- 
phuric acid  allowed  to  enter  the  tissues,  ammonia  being  set  free. 
If  we  assume,  for  instance,  that  the  tissue  alkali  is  comparable  to 
a  strong  base  like  sodium  hydroxid  or  barium  oxid,  the  ammonia 
introduced  in  combination  with  the  sulphuric  acid  will  be  absolutely 
unable  to  prevent  the  poisoning;  the  weak  base  will  be  forced  out 
of  the  salt  and  replaced  by  the  stronger  base.  In  general  we  must 
assume  that  the  antitoxin  possesses  a  higher  affinity  to  the  toxin 
than  do  the  tissue  receptors,  for  only  on  this  assumption  can  we 
explain  the  protective  action  of  the  antitoxin.  Numerous  phenomena, 
however,  indicate  that  the  affinity  of  the  tissue  receptors  can  become 
increased.  I  had  reached  these  conclusions  long  before  the  pub- 
lication of  my  theory,  which  as  many  know  I  formulated  years  before 
it  was  published.  The  cause  of  this  long  delay  was  the  phenomenon 
of  hypersusceptibility,  i.e.,  the  peculiar  fact  that  immunized  ani- 
mals, despite  a  colossal  excess  of  antitoxin,  succumb  to  the  action 
of  the  poison.  The  first  light  on  this  subject  was  the  study  of  Donitz, 
in  which  it  was  shown  that  the  poison  shortly  after  its  union  with 
the  tissues  is  but  loosely  bound.  In  the  course  of  a  few  hours  the 
union  becomes  firmer  and  firmer  so  that  after  a  certain  time,  which 
may  vary  from  a  few  minutes  to  six  hours,  according  to  the  dose, 
the  poison  can  no  longer  be  abstracted  from  the  tissues  by  the  anti- 
toxin. This  fact  seemed  to  indicate  that  under  the  influence  of 
the  poisoning  the  affinity  of  the  tissue  receptors  gradually  becomes 
Increased  and  that  when  a  certain  point  is  reached  a  cure  by  means 
of  antitoxin  is  impossible.  This,  however,  furnished  me  with  an 
explanation  of  hypersusceptibility  and  removed  the  obstacle  which 
had  kept  me  from  publishing  my  theory. 


522  COLLECTED  STUDIES  IN  IMMUNITY. 

I  should  also  like  to  mention  that  Kretz,1  many  years  later  and 
entirely  independent  of  me,  reached  exactly  the  same  conclusions 
as  I  had.  His  very  interesting  study  was  based  on  experiments 
with  diphtheria-immune  horses.  Following  his  usual  tactics,  Gruber 
will,  of  course,  draw  the  conclusion  that  the  increase  in  the  tissues 
affinity,  since  it  agrees  with  my  theory,  cannot  really  occur,  and 
he  will  therefore  regard  the  entire  subject  as  utterly  fallacious  and 
best  not  discussed.  The  unprejudiced  observer,  however,  need 
hardly  be  told  that  it  is  impossible  for  chemical  groups  attached 
to  living  protoplasm  to  maintain  their  affinity  unchanged  as  though 
they  were  made  of  stone;  especially  is  this  true  if  we  consider  the 
varying  function  of  the  protoplasm. 

Let  us  take  anilin  as  an  example,  and  determine  the  combining 
heat  of  the  NH2  group  for  a  certain  acid.  We  shall  then  find  that 
nearly  all  substitutions  of  the  benzol  nucleus,  as,  for  instance,  the 
introduction  of  an  amido  group,  a  nitro  group,  a  sulfo  group,  etc., 
markedly  change  the  affinity  either  positively  or  negatively.  Thus 
even  the  introduction  of  what  is  conceivably  the  most  indifferent 
group,  the  methyl  radical  causes  a  distinct  and  marked  diminution 
of  the  combining  heat.  Under  these  circumstances  any  one  who 
thinks  chemically  would  consider  it  peculiar  if  a  change  in  the  affinity 
of  the  cell  constituents  were  to  be  regarded  as  something  absolutely 
inconceivable  and  beyond  the  pale  of  discussion. 

Since  Gruber  has  given  only  that  part  of  Madsen  and  Dreyer's 
experiments  which  fits  into  his  polemic,  it  will  be  necessary  for  me 
to  supplement  this  with  some  additional  data  from  their  study. 

These  authors  employed  a  diphtheria  poison  of  which  the  fatal 
dose  for  a  guinea-pig  of  250  grammes  was  0.009,  and  for  rabbits 
of  1200-1600  grammes,  0.0076.  Calculated  per  kilo  this  shows 
that  the  rabbits  were  about  six  times  as  susceptible  as  guinea-pigs. 
The  L0  dose,  i.e.,  that  amount  of  poison,  which  is  just  completely 
neutralized  by  one  immune  unit,  was  0.6  cc.  for  guinea-pigs.  Right 
here  I  must  emphasize  that  the  L0  dose,  as  I  conceive  it,  refers  exclu- 
sively to  guinea-pigs,  since  according  to  my  experiences  this  is  the 
only  animal  in  which,  thanks  to  the  peculiar  susceptibility,  the  con- 
stants of  the  poison  can  accurately  be  determined.  In  the  serum 
mixture  L0  all  the  constituents  of  the  poison,  toxin,  and  toxon  are 
completely  neutralized,  so  that  not  only  the  single  amount  but  also 

1  Zeitsch.  f.  Heilk.,  Vol.  23,  1902. 


TOXIN  AND   ANTITOXIN  523 

high  multiples  of  this  can  be  injected  into  guinea-pigs  without  causing 
a  trace  of  local  or  general  reaction.  If  the  same  amount  of  poison, 

1  f\*7 

0.6  cc.,  was  mixed  with  —  I.  E.  instead  of  with  one  I.  E.  it  was 

found  that  the  toxin  fraction  had  practically  been  completely  neu- 
tralized, leaving  only  the  toxons,  characterized  by  the  develop- 
ment of  paralyses.  Just  in  this  poison  Madsen  and  Dreyer  have 
shown  that  the  difference  between  toxin  and  toxon  is  qualitative 
and  not  quantitative.  They  found  that  mixtures  of  poison  and 
antitoxin,  which  were  near  the  limit  of  toxin  neutralization,  showed 
only  toxon  action  when  given  in  small  doses,  whereas  when  the  mix- 
ture was  increased  tenfold,  death  occurred  from  toxin.1 

//,  however,  the  quantity  of  antitoxin  was  also  slightly  increased, 
even  the  tenfold  multiple  showed  only  toxon  action.  From  these  data 
we  see  that  the  poison  consisted  of  about  167  units  toxin-toxoid 
and  33  units  toxon. 

This  same  poison  was  subjected  to  a  thorough  investigation  on 
rabbits  by  Dreyer  and  Madsen  and  gave  the  following  results:  If 
0.6  cc.  poison  are  mixed  with  one  I.  E.,  it  will  be  found  that  this 
mixture,  which  represents  the  L0  dose  for  guinea-pigs,  is  still  highly 
toxic  for  rabbits.  In  order  to  render  this  amount  of  poison  com- 
pletely innocuous  for  rabbits  it  is  necessary  to  add  more  antitoxin; 

240 

as  a  matter  of  fact  it  requires  -  ^  IVE.     Their  statements  concern- 

zoo 

ing  the  behavior  of  mixtures  between  these  two  limits  are  also  very 

210 

interesting.     A  mixture  of  0.6  cc.  poison  +  —  I.  E.  given  to  a  rabbit 

gives  rise  to  paralytic  phenomena  appearing  on  the  fifteenth  day 
and  ending  fatally  on  the  twenty-second  day.  Even  a  mixture  of 

232 

the  same  dose  of  poison  with  — —  I.  E.  produced  paralysis  com- 
mencing on  the  sixteenth  day  and  continuing  for  several  weeks. 
In  view  of  the  importance  of  these  facts  for  the  conception  of  a  plu- 
rality of  poisons,  I  cannot  pass  on  without  discussing  them  more 
fully.  According  to  our  definition  of  the  L0  dose,  such  over-neu- 

1  The  explanation  of  this  is  that  the  toxon  determination  by  means  of  1  I.  E. 
naturally  cannot  be  an  absolutely  exact  one,  small  residual  amounts  of  toxin, 
«.g.,  1/10  lethal  dose,  readily  being  overlooked.  If,  however,  an  appropriate 
multiple,  say  ten  times  this  mixture,  be  injected,  this  will  contain  ten  times 
1/10  fatal  dose. 


524  COLLECTED  STUDIES  IN  IMMUNITY 

232\ 
like  the  mixture        r     possess  a  considerable 


excess  of  antitoxin,  are  absolutely  innocuous  for  guinea-pigs  and 
can  be  injected  in  any  desired  quantity.  In  fact,  owing  to  the  excess 
of  antitoxin,  such  mixtures  furnish  the  animal  with  passive  immunity 
and  protect  it,  provided  suitable  amounts  have  been  injected,  against 
diphtheria  poison  and  diphtheria  bacilli.  If  such  mixtures,  how- 
ever, are  still  toxic  for  rabbits,  only  one  possibility  remains,  namely, 
that  the  diphtheria  poison  in  question  contains  a  substance  which 
is  non-toxic  for  guinea-pigs,  but  still  toxic  for  rabbits.  This  is  my 
toxonoid.1 

So  far  as  the  behavior  of  partially  neutralized  mixtures  is  con- 
cerned, the  investigations  of  the  two  authors  show  that  mixtures 
which  exert  only  toxon  effects  on  guinea-pigs  cause  death  and  symp- 
toms of  diphtheria  poisoning  in  rabbits.  In  my  opinion  the  phe- 
nomenon described  can  best  be  explained  by  the  assumption  that 
at  least  three  varieties  of  poison  are  to  be  distinguished,  possessing 
different  affinities  and  different  actions.  Such  an  assumption,  I 
believe,  will  best  harmonize  the  actual  facts.  These  poisons  are: 

1.  Toxin,  possessing  the  greatest  affinity,  kills  rabbits  and  guinea- 
pigs  acutely,  but  is  much  more  toxic  for  the  former. 

2.  Toxon,  killing  rabbits  acutely  and  guinea-pigs  with  paralytic 
symptoms. 

3.  Toxonoids,  producing   paralyses  in  rabbits  but   innocuous  for 
guinea-pigs. 

That  all  these  poisons  act  more  powerfully  on  rabbits  than  on 
guinea-pigs  is  explained  by  the  absolute  higher  susceptibility  of 
these  animals.  So  far  as  the  behavior  of  the  toxonoids  is  concerned, 
in  which  enormous  differences  in  rabbits  and  guinea-pigs  are  mani- 
fested, such  behavior  finds  numerous  analogies  in  toxicology,  espe- 
cially in  the  study  of  toxins.  Thus  heroin,  an  acetyl  derivative 

1  Almost  at  the  outset  of  my  investigations  and  long  prior  to  Madsen  and 
Dreyer  I  obtained  results  entirely  similar  to  these.  My  unpublished  but  very 
extensive  studies  showed  that  this  property  is  not  possessed  by  all  diphtheria 
poisons,  for  I  also  encountered  poisons  in  which  the  L0  dose  was  exactly  the 
same  in  guinea-pigs  and  rabbits.  This  fact  controverts  the  assumption  that 
perhaps  the  described  phenomenon  is  due  to  an  incomplete  neutralization, 
such  as  Arrhenius  and  Madsen  have  demonstrated  in  the  union  of  boric  acid 
and  ammonia,  and  in  that  of  tetanolysin  and  antilysin.  If  this  were  the  case 
one  would  expect  the  phenomenon  to  be  present  in  all  diphtheria  poisons  to 
the  same  extent,  and  this  is  not  the  case. 


TOXIN  AND  ANTITOXIN  525 

of  morphine,  is  far  less  toxic  for  rabbits  than  is  morphine;  for  asses 
on  the  other  hand  it  is  far  more  toxic  than  the  latter  substance.  In 
the  case  of  toxins  v.  Behring  long  ago  showed  that  for  different 
species  of  animals  certain  toxins  are  very  differently  affected  by 
trichloriodine.  As  I  suggested  in  my  address  at  the  International 
Medical  Congress  in  Paris  we  are  evidently  dealing  here  with  incom- 
plete toxoids,  i.e.,  with  toxoids  whose  toxophore  complex  is  not 
yet  completely  destroyed.  Portions  of  this  complex  still  left  to 
the  poison  possess  a  high  toxicity  for  one  species  of  animal  and  little 
or  no  toxicity  for  another.  The  toxophore  groups  of  the  tetanus 
poisons  mentioned  above  (Tizzoni  and  v.  Behring)  afford  a  sufficient 
analogy. 

A  consideration  of  these  facts  will  show  that  Gruber's  statement, 
that  the  facts  observed  by  Madsen  and  Dreyer  reduce  my  theory 
to  an  absurdity,  is  absolutely  incorrect.  On  the  contrary,  I  may 
say  that  the  facts  brought  out  by  these  authors  are  most  readily 
explained  on  the  basis  of  my  theory. 


I  shall  now  take  up  Gruber's  recent  experiments.  These  were 
first  published  in  the  Wiener  klin.  Wochenschrift l  in  a  form  strongly 
suggestive  of  the  comic  supplement  of  a  newspaper. 

The  discussion  takes  the  form  of  a  letter  purporting  to  be  written 
by  a  certain  "  Phantasus,"  and  is  really  very  cleverly  conceived. 
Only  I  would  protest  against  publications  of  this  sort  appearing  in 
the  columns  of  a  scientific  journal. 

TWTO  series  of  experiments  come  into  question.  The  first  series 
is  so  curious  that  I  have  not  felt  any  desire  to  repeat  the  experi- 
ments. These  deal  (a)  with  the  property  of  sulphuric  acid  to  act 
as  a  poison  on  cane  sugar,  and  (6)  with  the  antitoxic  action  which 
water  exerts  on  this  property.  Any  one  with  even  the  faintest  knowl- 
edge of  chemical  processes  knows  that  the  sulphuric  acid  as  such 
is  not  deprived  of  this  poisonous  action  by  water;  this  is  effected 
only  by  an  alkali  which,  by  forming  a  salt,  neutralizes  the  acid.  I 
am  able  to  furnish  an  additional  case  which  shows  the  "  detoxitizing  " 
effect  of  water.  A  highly  concentrated  sulphuric  acid,  containing 
considerable  anhydride,  acts  destructively  on  iron.  If  H2O  is  added 
until  the  solution  contains  the  monohydrate  it  will  be  found  that 
the  addition  of  the  water  has  reduced  this  capacity  to  attack  iron 

1  Wiener  klm.  Wochenschr.,  No.  27,  1903. 


526  COLLECTED  STUDIES  IN    IMMUNITY. 

to  practically  zero.  In  this  case  then,  just  as  Gruber  states,  the 
water  has  acted  as  an  antitoxin.  On  the  addition  of  more  water 
to  the  mixture,  however,  the  iron  is  again  attacked.  In  fact  the 
more  water  now  added  the  stronger  becomes  this  action.  We  thus 
obtain  the  curious  result  that  in  small  doses  water  acts  as  antitoxin* 
while  in  large  doses  it  increases  the  action  of  the  poison,  surely  an 
interesting  problem  for  Dr.  Phantasus! 

This  is  merely  one  of  the  special  instances  of  the  fact  thus  far 
unexplained,  that  the  different  hydrates  of  sulphuric  acid,  or  their 
mixtures,  manifest  a  most  extraordinary  variation  of  properties- 
I  may  refer  the  reader  to  the  minute  and  fundamental  study  of 
Knietsch,1  in  which  the  variations  of  the  properties  of  sulphuric 
acid  at  different  concentrations  have  been  represented  in  the  form 
of  a  curve  for  many  of  these  properties,  thus  specific  heat,  electric 
resistance,  boiling  point,  vapor  tension,  viscosity,  capillarity,  action 
on  iron,  etc.  A  glance  at  this  chart  gives  one  the  impression  of 
chaos,  and  at  once  shows  that  on  these  complicated  problems  only 
deep  studies  can  lead  to  any  results,  and  that  the  ten-minute  experi- 
ments made  by  Phantasus-Gruber-Pirquet  are  absolutely  worthless. 
This  is  especially  true  in  Gruber's  case,  which  deals  with  an  obscure 
reaction  in  which  oxidation,  abstraction  of  water,  cleavage  and  sul- 
phurization  take  part.  Hence  I  deny  that  crude  experiments  of 
this  kind  can  be  used  to  gain  an  insight  into  such  an  entirely  different 
subject,  or  that  the  conditions  there  observed  can  even  be  com- 
pared to  the  minutely  differentiated  processes  of  toxin-antitoxin 
combination. 

We  shall  next  take  up  Gruber's  experiments  which  deal  with 
the  hsemolytic  action  of  water,  since  to  persons  at  a  distance  these 
might  give  the  impression  that  they  really  have  something  in  com- 
mon with  studies  in  hsemolytic  toxins.  The  experiments  are  sup- 
posed to  show  that  water  is  composed  of  an  infinite  number  of  differ- 
ent poisons.  Let  us  listen  to  Gruber  for  a  moment: 

"  Pure  water  exercises  a  very  great  osmotic  pressure  on  red 
blood-cells,  leading  to  their  swelling  and  to  the  escape  of  haemoglobin. 
Hence  water  is  a  toxin  for  the  erythrocytes,  salt  is  an  antitoxin. 
When  successive  amounts  of  salt  are  added  to  the  water  this  toxicity 
is  gradually  lost,  for  the  affinity  of  the  water,  and  with  it  the  osmotic 
pressure,  is  thus  gradually  decreased." 

1  Bericht  d.  deutsch.  chem.  Gesellschaft,  1901,  page  4069. 


TOXIN   AND  ANTITOXIN  527 

We  see  therefore  that  Gruber-Pirquet  assume  that  pure  water 
possesses  a  high  osmotic  pressure  and  that  salt  diminishes  this.  The 
very  foundation  of  the  doctrine  of  osmotic  tension,  however,  is  the 
fact  that  water  as  such  possesses  NO  osmotic  pressure,  and  that 
such  pressure  is  produced  by  salts  dissolved  in  the  -water.  I  can- 
not refrain  from  pointing  out  this  woful  ignorance  of  the  most  ele- 
mentary principles  on  the  part  of  authors  who  do  not  hesitate  to 
accuse  me  of  "  complete  lack  of  insight  into  chemistry/'  although 
for  years  I  have  endeavored,  and  not  unsuccessfully,  to  apply  the 
great  discoveries  in  chemistry  to  medicine. 

The  solution  of  erythrocytes  by  means  of  water  is  one  of  the 
best  studied  subjects  in  medicine.  It  is  generally  recognized  that 
the  water  as  such  is  no  poison  whatever,  but  that  its  action  is  due 
to  the  fact  that  water  abstracts  the  salts  and  other  soluble  substances 
from  all  living  cells,  including,  of  course,  the  red  blood-cells.  These 
substances  are  abstracted  in  such  considerable  amounts  that  this 
alone  suffices  to  bring  about  the  death  of  the  cell.  The  swelling 
of  the  red  blood-cells  is  due  to  the  penetration  of  water  and  this 
again  depends  on  the  permeability  of  the  limiting  membrane  on 
the  one  hand  and  the  power  of  the  water  to  abstract  water  on  the 
other. 

With  the  same  right  that  Gruber  regards  water  as  a  poison  one 
could  call  nitrogen  a  poison  and  oxygen  as  the  counter  poison  for 
the  nitrogen,  for  animals  die  in  pure  nitrogen,  but  live  if  oxygen  is 
added.  At  any  rate  nitrogen  poison  can  be  recommended  to  Dr.. 
Phantasus  for  extended  study.  Perhaps  some  day  he  will  also  work 
out  its  spectrum  for  us. 

Despite  the  fact  that  the  premises  from  which  their  experiment 
proceeds  are  based  on  a  complete  misconception  of  the  idea  of  poison, 
I  have  repeated  the  experiments  of  Gruber  and  Pirquet.  The  results 
show  that  their  statements  concerning  the  experiment  are  entirely 
incorrect.  I  first  determined  the  concentration  of  salt  and  of  sugar, 
in  which  the  ox  blood-cells  remained  completely  intact;  for  NaCl 
this  was  found  to  be  0.63%,  for  cane  sugar  6.4%.  By  diluting 
with  water,  various  degrees  of  this  isotonicity  (1/10,  2/10,  etc.) 
were  produced.  Each  tube  contained  altogether  2  cc.  of  fluid  and 
one  drop  of  defibrinated  ox  blood.  The  result  is  shown  in  the  form 
of  a  "  spectrum,"  which  may  be  compared  to  that  obtained  by~ 
Gruber  in  his  experiments. 

This  comparison  shows  us  that  Gruber's  experiments  are  abso- 


528 


COLLECTED   STUDIES   IN-  IMMUNITY 


lutely  incorrect,  and  that  they  contradict  all  that  is  thus  far  known 
concerning  solution  of  the  red  blood-cells.  Gruber  states  that  in 
a  1/10  isotonic  solution,  one  containing  about  0.07%  NaCl,  about 
one-fifth  of  the  blood-cells  remain  undissolved.  All  other  authors, 
however,  have  found  that  even  in  a  solution  of  0.3%  NaCl,  the 
blood-cells  of  all  warm-blooded  animals  are  still  completely  dis- 
solved, so  that  the  solution  appears  uniformly  laky,  and  microscopical 
examination  shows  not  even  a  trace  of  red-blood  corpuscles.  In 
Gruber's  spectrum,  however,  we  find  that  with  this  percentage  more 
than  half  of  the  blood-cells  remain  undissolved.  This  indicates 
that  in  Gruber's  experiments  the  grossest  sort  of  errors  abound. 


With  Salt 

Decrease  of 
Hamolyse  in  Percent 

30 


20 


15 


With  Sugar 

Decrease  of 
Hamolyse  in  Percent 

30 


— I 


25 


20 


15 


10 


^ 


m_ 

I     I — V~^\ 


%    9/io 


Isotonicity  Isotonicity 

FIG.  1. — "  Poison  spectrum  "  of  water  according  to  Gruber. 

What  can  we  deduce  from  these  spectra?  The  fact  that  a  cer- 
tain amount  of  NaCl  can  be  added  to  the  "  poisonous  "  water  with- 
out inhibiting  haemolysis,  would  lead  authors  holding  Gruber's  views 
to  conclude  that  this  "  poisonous  "  water  contains  a  prototoxoid 
whose  neutralization  has  no  effect  whatever  on  the  toxic  action. 
A  single  glance  at  the  detailed  literature  on  this  subject  should,  how- 
ever, have  convinced  these  authors  that  their  curve,  as  such,  has 
nothing  whatever  to  do  with  toxic  actions,  but  is  merely  the  expres- 
sion of  the  specific  differences  in  the  red  blood-cells.  It  is  well  known 


TOXIN   AND  ANTITOXIN 


529 


that  the  blood  represents  a  mixture  of  cells  of  various  ages,  and  it 
is  not  at  all  surprising,  therefore,  that  these  should  behave  differently 
toward  different  injurious  influences.  We  are  here  dealing  with  a 
property  of  the  erythrocyte's  protoplasm,  which  protoplasm  will  possess 
a  different  degree  of  vulnerability  according  to  its  age.  Are  Gruber- 
Pirquet  entirely  unaware  that  an  important  and  much-employed 
procedure  for  determining  the  resistance  of  the  blood  rests  on  just 


With  Salt 


With  Sugar 


Decrease 


30 


15 


10 


No  Decrease 


Decrease 
35 


30 


20 


15 


10 


No  Decrease 


Isotonicity  Isotonicity 

FIG.  2. — "  Poison  spectrum"  of  water  according  to  Ehrlich. 

this  principle?  Every  text-book  on  hsematology  teaches  that  we 
distinguish  blood-cells  of  maximum,  minimum,  and  intermediate 
resistance,  and  that  the  extent  of  resistance  is  merely  the  difference 
between  the  maximum  and  minimum. 

Instead  of  this,  however,  Gruber  feels  compelled  to  draw  from 
his  curves  conclusions  having  such  far-reaching  consequences  as, 
for  example,  that  water  is  full  of  poisons,  of  haptophore  and  toxo- 
phore  groups,  etc.  But  if  he  believes  that  this  proves  the  folly  of 
my  conception  of  toxin  neutralization,  so  much  the  worse  for  him 
and  his  authority  Phantasus. 


530  COLLECTED  STUDIES  IN   IMMUNITY. 

If  one  conducts  experiments  that  have  nothing  to  do  with  the 
problem  under  discussion,  further,  if  the  method  of  these  experi- 
ments is  grossly  at  fault,  and  it,  finally,  the  results  thus  obtained 
are  given  an  utterly  false  interpretation,  it  is  not  surprising  that 
the  most  fantastic  results  are  obtained. 

Finally  Gruber  describes  one  more  experiment  which  he  illus- 
trates by  means  of  a  curve.  According  to  him  this  too  demonstrates 
that  my  theory  is  untenable.  The  experiment  shows  that  the  haemol- 
ysis of  ox  blood,  by  means  of  a  certain  quantity  of  specific  hsemolytic 
serum  within  half  an  hour,  is  dependent  on  the  dilution.  I  need 
hardly  remind  my  readers  that  1  have  always  laid  stress  on  the  chemi- 
cal nature  of  the  toxin  and  antitoxin  combination.  I  can  assure 
them  that  the  factor  of  the  degree  of  concentration  has  ever  been 
sufficiently  regarded.  If  Gruber  will  refer  to  my  first  study  on  this 
subject,  "  Die  Werthbemessung  des  Diphtheneheilserums,"  he  will 
find  the  statement:  "  that  the  union  of  poison  and  antibody  pro- 
ceeds much  more  rapidly  in  concentrated  than  in  dilute  solutions/' 
and  further  also  "  that  heat  hastens  the  union  and  cold  retards 
the  same." 

The  behavior  which  Gruber  describes  is  all  the  less  surprising 
since  he  is  dealing  with  a  complex  process  depending  on  the  action 
ot  the  amboceptor-complement  combination.  How  readily  this 
combination  is  dissociated  has  repeatedly  been  pointed  out  by  usp 
Perhaps  Gruber  thinks  that  this  experiment  is  new  to  me;  every 
one  versed  in  the  subject,  however,  knows  that  we  are  here  deal- 
ing with  the  most  commonplace  phenomena,  with  which  every  beginner 
is  well  acquainted.  I  should  like  to  point  out,  however,  that  this 
phenomenon,  namely,  that  dilution  with  water  inhibits  the  action 
of  haemolysins,  is  not  at  all  constant.  On  the  contrary  it  is  limited 
to  those  cases  in  which  the  affinity  between  amboceptor  and  cell, 
or  between  amboceptor  and  complement  is  relatively  slight.  If 
one  employs  poisons  in  which  the  affinity  between  receptor  and 
cell  is  great  it  will  be  found  that  within  the  limits  mentioned  the 
addition  of  water  is  practically  without  effect.  Thus,  in  working 
with  cobra  venom,  I  found  that  a  given  quantity  of  this  poison 
exerted  exactly  the  same  effect  whether  the  volume  of  water  used 
was  1  or  15. 

It  would  lead  us  too  far  to  enter  into  all  the  distortions  and  mis- 
conceptions contained  in  Gruber's  polemic.  To  do  this  would  require 
almost  a  complete  reprint  of  all  my  articles,  as  well  as  of  many  others 


TOXIN  AND  ANTITOXIN.  531 

emanating  from  the  Institute — with  all  of  which  Gruber  seems  quite 
unfamiliar.  I  shall  content  myself  therefore  with  a  brief  discussion 
of  Gruber's  conclusions.  Gruber  states: 

1.  "  There  is  no  warrant  for  assuming  that  the  bacterial  toxie 
solutions  contain  a  number  of  poisons  possessing  qualitatively  simi- 
lar actions  but  differing  in  intensity  and  in  their  affinity  to  the  anti- 
toxin." 

In  the  preceding  pages  I  have  conclusively  shown  that  his  view 
cannot  be  harmonized  with  the  actual  facts:  But  even  a  priori 
there  is  no  reason  to  assume  that  bacterial  cells  always  produce- 
only  a  single  poisonous  metabolic  product.  Thus,  to  mention  only 
a  few  examples,  we  know  that  cinchona  bark  contains  about  twenty- 
different  alkaloids,  opium  about  the  same  number;  Flexner  and 
Noguchi's  researches  show  that  snake  venom  contains  at  least  four 
different  poisons  (haemotoxin,  leucotoxin,  neurotoxin,  endothelio- 
toxin),  and  the  yeast  cell,  we  know,  contains  a  number  of  different 
ferments.  Furthermore,  1  may  again  call  attention  to  the  fact  that 
the  secretion  of  tetanus  bacilli  contains  four  distinct  poisons,  namely, 
two  varieties  of  tetanospasmin,  my  tetanolysin,  and  the  poison 
which,  according  to  Tizzoni,  causes  the  cachexia.  So  far  as  diphtheria 
poison  is  concerned  the  reader  is  referred  to  my  previous  statements. 
My  assumption  of  the  existence  of  at  least  two  poisons,  toxins,  and 
toxons,  is  borne  out  by  the  clincal  observation  that  in  certain  epi- 
demics there  is  a  large  percentage  of  paralyses.1 

2.  "  There  is  no  reason  for  assuming  that  the  mode  of  action  of 
the  toxins  is  absolutely  unlike  that  of  other  organic  poisons." 

Nevertheless,  the  fact  remains  that  the  principal  characteristic 
of  the  toxins,  namely,  the  production  of  antibodies,  does  differentiate 
them  from  all  other  poisons,  Gruber  to  the  contrary  notwithstand- 
ing. Two  years  ago  Gruber  could  have  found  an  ally  in  Pohl,  who 


1  In  animal  experiments  as  a  rule,  the  toxons  do  not  manifest  themselves 
until  the  toxins  (which  possess  a  greater  affinity)  have  been  neutralized  by 
the  antitoxin.  Dreyer  and  Madsen,  however,  have  described  a  diphtheria 
poison  (Festskrift,  Kopenhagen,  1902),  in  which  the  toxons  could  be  demon- 
strated even  by  the  injection  of  sublethal  doses,  the  injections  being  followed 
by  paralytic  phenomena.  In  view  of  the  constants  of  this  poison,  as  they  were 
determined  by  Dreyer  and  Madsen,  this  behavior  is  not  at  all  surprising,  for 
while  old  diphtheria  bouillons  ordinarily  contain  about  33  toxon  equivalents 
to  167  toxin  equivalents,  this  poison  contained  about  500  toxon  equivalents 
for  that  amount  of  toxin. 


532  COLLECTED  STUDIES   IN   IMMUNITY 

had  apparently  succeeded  in  immunizing  against  solanin.  Since 
then,  however,  the  researches  of  Bashford  l  and  of  Besredka9  have 
shown  that  it  is  impossible  to  produce  antibodies  against  either 
solanin  or  saponin.  Pohl  himself  no  longer  maintains  the  existence 
of  a  specific  antisolanin.  Of  the  various  poisons,  which  seemed 
to  promise  the  best  for  successful  immunization,  morphine  should 
be  mentioned  first.  Recently  Hirschlaff  3  claimed  actually  to  have 
produced  an  antimorphine  serum.  Morgenroth,4  however,  was  able 
to  show  that  the  results  obtained  by  Hirschlaff  were  merely  apparent, 
not  real,  and  that  they  depended  on  the  fact  that  the  doses  of  poi- 
son employed  by  Hirschlaff  were  not  surely  fatal,  especially  owing 
to  the  increased  resistance  of  the  animal  following  the  serum 
injection.  Hence  the  statement  still  holds  true  that  all  poisons 
chemically  well  defined  do  not  possess  the  property  of  producing 
antitoxins. 

So  far  as  other  differences  between  ordinary  poisons  and  toxins 
are  concerned,  I  may  refer  particularly  to  my  detailed  articles  in 
von  Leydens  Festschrift  5  and  to  the  excellent  monograph  by  Over- 
ton.6  From  these  it  will  be  seen  that  the  action  of  the  chemically 
defined  poisons,  alkaloids,  glucosides,  etc.,  on  parenchyma  is  the 
result  of  a  solid  solution  or  of  a  loose  salt  formation.  In  accordance 
with  the  loose  character  of  the  combination,  the  action  of  these 
poisons  is  a  transitory  one.  The  firm  union  and  prolonged  action 
peculiar  to  the  toxins  is  entirely  absent.  Besides  this  the  period 
of  incubation  is  wanting  in  most  ordinary  poisons,  although  there 
are  a  few  exceptions  like  arsenic,  phosphorus,  tartrate  of  tin  and 
sodium,  and  vinylamin.  In  the  toxins,  on  the  other  hand,  a  period 
of  incubation  is  the  rule. 

Entirely  in  accordance  with  the  views  of  Emil  Fischer  concern- 
ing ferments,  I  have  ascribed  the  specific  combining  processes  of 
toxins  to  certain  stereochemical  groups  of  atoms  (haptophore  groups). 
These  unite  only  with  such  other  atomic  groups  which  fit  to  them 
as  does  a  key  to  a  lock.  The  ordinary  chemical  groups  of  organic 
chemistry  possess  affinities  for  a  large  number  of  other  groups.  Thus 


1  Archives  Internationales  de   Pharmacodynamics,   Vols.   8  and  9. 

2  See  Metchnikoff,  L'Immunite,  Paris,  1901. 

3  Berliner  klin.  Wochenschrift    1902. 

4  Ibid.,  1903,  No.  21. 

6  Von   Leydens  Festschrift.  August  Hirschwald,  Berlin,  1902. 
8  Studien  iaber  die  Narkose,  Jena    1901. 


TOXIN   AND  ANTITOXIN  533 

the  aldehyde  group  can  unite  with  amido  groups,  hydrazin  groups, 
methylen  groups,  etc.  In  this  group  therefore  the  combining  prop- 
erty is  not  specifically  limited,  but  extends  to  a  large  number  of 
combinations.  On  the  other  hand  the  one  characteristic  of  toxins 
and  ferments  is  just  this  specific  combining  property. 

3.  "  The  transformation  of  toxins  into  non-poisonous  combina- 
tions (toxoids),  possessing  the  same  affinity  for  the  antitoxin  is  pos- 
sible, but  has  not  been  definitely  proven." 

I  have  already  clearly  shown  that  the  doctrine  of  toxoids,  now 
generally  accepted,  is  one  of  the  best-established  foundations  in  the 
entire  subject  of  immunity.  However,  with  critics  like  Gruber, 
who  blindly  condemn  the  views  of  others,  one  ought  to  be  satisfied 
if  they  recognize  at  least  a  possibility. 

4.  "  Toxin    and    antitoxin    have    feeble    chemical    affinities    and 
therefore  unite  with  one  another  to  form  dissociable  combinations  or 
perhaps  molecular  combinations  in  varying  proportions.     These  con- 
ditions explain  the  long  incubation  of  the  poisonous  action  and  other 
marked  phenomena." 

To  be  sure  the  affinity  between  toxin  and  antitoxin  may  in  some 
instances  be  a  feeble  one,  but  this  is  by  no  means  always  the  case. 
The  affinity  between  tetanus  toxin  and  antitoxin  is  slight,  and  so 
is  that  between  complement  and  amboceptor.  On  the  other  hand, 
however,  there  are  poisons,  such  as  diphtheria  toxin  and  snake  venom, 
in  which  the  reaction  proceeds  under  strong  affinities,  so  that  the 
process  of  neutralization  takes  the  course  of  a  straight  line  and  not 
of  a  curve. 

Gruber's  statements  might  also  give  one  the  impression  that 
he  is  the  first  to  introduce  dissociation  as  an  explanation  of  some 
of  the  phenomena  in  immunity.  I  have  always  emphasized  the 
fact  that  amboceptor  and  complement  are  loosely  bound,  uniting 
at  high  temperatures,  but  dissociating  at  low  temperatures.1  But 
this  is  all  wrong  according  to  Gruber,1  for  a  year  and  a  half  ago  he 


1  I  shall  cite  a  passage  from  Ehrlich  and  Morgenroth's  First  Communi- 
cation Concerning  Hsemolysins  (see  page  7  of  this  volume),  a  passage  which 
Wechsberg  has  already  called  to  Gruber's  attention  (Wiener  klin.  Wochenschr. 
1901,  No.  51).  "This  experiment  clearly  shows  that  under  the  conditions 
present  complement  and  immune  body  exist  in  the  serum  independently  of 
one  another  ";  further  also,  "  under  certain  circumstances  the  immune  body 
enters  into  a  loose  chemical  union  with  the  complement,  one  which  is  easily 
dissociated."  In  view  of  this  I  cannot  understand  why  Gruber  still  main- 


534  COLLECTED  STUDIES  IN   IMMUNITY 

laid  down  the  dictum,  "  There  is  no  dissociation  by  means  of  cold." 
It  seems  not  to  have  mattered  to  him  that  his  statement  is  opposed 
to  even  the  most  elementary  principles  of  chemistry. 

As  a  matter  of  fact  we  have  always  paid  due  attention  to  disso- 
ciation and  to  the  reversibility  of  the  reactions.  I  should  like  to 
call  Gruber's  attention  to  the  fact  that  the  sentence:  "  In  the  union 
of  the  amboceptors  we  are  dealing  with  a  reversible  process  "  occurs 
in  one  of  Morgenroth's  studies  2  from  this  Institute.  Further  than 
this  such  questions  do  not  affect  the  Side-chain  Theory,  as  such.  The 
whole  discussion  is  evidently  designed  to  hide  the  fact  that  Gruber's 
position  is  really  based  on  my  theory. 

So  far  as  the  mode  of  action  of  the  toxins  is  concerned, 
Gruber's  standpoint  and  mine  are  essentially  the  same.  Thus 
Gruber  states  that:  *'  All  poisons  .must  be  'anchored'  by  the 
cells  and  the  anchoring  group  of  atoms  is  probably  always  different 
from  that  group  which  gives  the  substance  its  toxicity."  I  spent 
many  years  in  establishing  this  view  and  it  is  now  everywhere  accepted 
as  axiomatic.  I  defy  Gruber  to  show  me  the  text-books  of  toxi- 
cology in  which,  previous  to  my  work,  this  conception  appears,  a 
conception  which  dominates  the  laws  of  the  distribution  and  action 
of  poisons.  If  he  should  again  refer  to  S.  FrankePs  book  3  I  can 
only  remark  that  while  the  account  of  my  views  is  very  admirable, 
it  is  nothing  more  than  a  resume  of  the  points  which  I  had  previously 
developed.  Perhaps  I  can  even  aid  Gruber's  memory  and  let  him 
speak  for  himself.  A  year  before  his  declaration  of  war  he  spoke 
of  "  the  brilliant  hypothesis  of  that  genius  Paul  Ehrlich,  the  greatest 
of  living  pathologists."  In  a  little  work4  published  at  that  time, 
and  quite  enthusiastic  over  my  theory  he  states:  "  According  to 
Ehrlich  only  such  substances  are  poisons  which  unite  chemically 
with  some  constituent  of  the  organism."  And  yet  this  same  Gruber 
to-day  says:  "  These  are  merely  new  words  for  what  has  long  been 
known." 

I  should  not  like  to  deprive  the  reader  of  hearing  still  another 

tains  that  ray  view  of  the  production  of  anticomplements,  according  to  which 
amboceptor  and  complement  are  firmly  united,  is  absolutely  incomprehensible. 

'  Munch,   med.   Wochenschr.    1901,   No.   48. 

'Ibid.,  1903. 

'  Die  Arzneimittelsynthese,  Berlin,  1901. 

*  Max  Gruber.  Neuere  Forschungen  iiber  erworbene  Immunitat,  Vienna, 
1900. 


TOXIN   AND  ANTITOXIN.  535 

authority  often  cited  by  Gruber,  namely  von  Behring.  Shortly  after 
my  theory  was  formulated  this  author  expressed  himself  as  follows:1 
"  It  seemed  about  hopeless  to  attempt  to  penetrate  these  mysteries, 
when  recently  Prof.  Ehrlich  published  a  theory  which  is  destined 
to  illuminate  even  this  subject." 

But  even  now  Gruber  does  not  doubt  "  that  the  toxins  are  very 
complex  bodies  and  that  the  toxic  action  is  connected  with  certain 
atomic  groups;  that  possibly  it  is  necessary  for  certain  atomic  groups 
to  be  present  so  that  the  poison  molecule  can  be  anchored  and  the 
toxicity  manifest  itself." 

One  will  at  once  ask  why  then  Gruber  attacks  my  theory  if  he 
is  satisfied  with  its  fundamental  principle,  namely,  the  assumption 
of  an  independent  haptophore  and  toxophore  group  in  the  poison 
molecule?  That  I  cannot  answer.  To  be  sure  further  along  one 
encounters  the  warning,  "  But  one  must  not  too  highly  personify 
these  different  atomic  groups,  and  think  of  this  entire  poisoning 
as  a  drama  with  four  long  intermissions  between  the  acts."  I  cannot 
see  what  is  to  be  gained  by  such  idle  talk. 

As  a  matter  of  fact  the  majority  of  infectious  diseases  as  well 
as  the  poisonings  do  proceed  in  three  phases,  and  these  have  always 
been  separated,  namely,  incubation,  the  disease  itself,  recovery. 
Hence  to  explain  these,  as  we  do,  through  the  independent  action 
of  toxophore  and  haptophore  groups  seems  the  most  natural  thing 
to  do.  It  is  strange  that  Gruber  should  now  speak  of  the  anchoring 
of  the  poison  by  the  elements  susceptible  .thereto  as  something  per- 
fectly obvious,  for  in  his  first  attack  he  laid  especial  emphasis  on 
"  his  being  the  first  to  furnish  the  important  demonstration  that 
the  specific  immune  substances  are  bound  by  the  bacteria."  How- 
ever, Gruber's  claim  cannot  be  allowed,  for  all  that  he  demonstrated 
was  that  the  agglutinins  are  used  up  in  the  reaction.  The  signifi- 
cance of  a  chemical  union,  however,  was  first  pointed  out  by  us. 
This  union,  as  Morgenroth's  studies  on  the  behavior  of  anchored 
amboceptors  show,  need  in  no  way  be  connected  with  toxic  action 
or  with  a  using  up  of  the  substance. 

Gruber's  statement  that  the  long  period  of  incubation  is  explained 
by  the  feeble  affinities  I  must  emphatically  deny.  The  studies  of 
Donitz  2  and  of  the  Heyman  school 3  show  that  the  injected  toxins 


1  Deutsche  med.  Wochenschr.  1898. 

'Ibid.,  1897. 

3  Decroly  et  Rouse,  Arch,  de  Internal,  de  Pharmaeodynamie,  Vol.   VI. 


536  COLLECTED  STUDIES  IN   IMMUNITY. 

disappear  from  the  circulation  in  a  few  minutes.  It  is  therefore 
idle  to  talk  of  a  slow  union  such  as  would  correspond  to  weak  affini- 
ties. But,  says  Gruber,  "it  is  impossible  to  understand  why  the 
toxophore  groups,  after  they  have  been  brought  into  proximity  to 
the  protoplasm,  do  not  at  once  commence  their  activity,  but  always 
stop  to  consider  the  matter  for  several  hours."  One  cannot  seriously 
discuss  the  subject  with  such  a  questioner.  Gruber  might  just  as  well 
ask  that  all  chemical  reactions  proceed  rapidly,  and  deny  the  possi- 
bility of  a  slow  reaction. 

The  slow  action  of  the  toxophore  group  is  not  at  all  remarkable, 
especially  in  the  domain  of  toxins.  This  is  particularly  true  if  we 
remember  that  with  certain  poisons  (e.g.  botulism  toxin),  one  part 
of  toxin  to  500  million  parts  of  body  weight  suffices  to  cause  death, 
and  that  the  rapidity  of  action  is  dependent  to  a  high  degree  on 
the  amount  of  the  active  substance. 

Is  Gruber  possibly  of  the 'opinion  that  in  the  paralysis  of  diph- 
theria, which  as  is  well  known  usually  develops  after  the  lapse  of 
weeks,  the  toxon  courses  about  free  for  twenty  days  or  more  before 
entering  the  tissues  and  then  suddenly  exerts  its  action?  To  the 
unprejudiced  critic  the  importance  of  the  separation  of  toxin  bind- 
ing and  toxin  action  for  the  proper  understanding  of  the  period  of 
incubation,  is  conclusively  demonstrated  by  Morgenroth's l  experi- 
ments with  .tetanus  in  frogs.  Courmont  and  Doyon,  as  is  well  known, 
discovered  that  the  frog  is  susceptible  to  tetanus  poison  only  at 
higher  temperatures,  and  not  when  the  animal  is  kept  cold.  Mor- 
genroth  was  able  to  show  that  at  low  temperatures  the  tetanus  poison 
is  bound,  but  exerts  no  toxic  action.  Frogs  are  injected  with  tetanus 
toxin  and  then  kept  on  ice  for  days.  If  then  they  are  subjected 
to  higher  temperatures,  it  will  be  found  that  they  behave  exactly 
as  if  they  had  just  been  inoculated.  And  yet  the  toxin  has  been 
bound  by  the  central  nervous  system  even  at  the  low  temperature; 
for  if  after  several  days  at  low  temperature  the  animal  be  injected 
with  an  amount  of  antitoxin,  even  much  more  than  sufficient  to 
neutralize  the  poison,  tetanus  will  still  develop  if  the  frog  is  subjected 
to  a  higher  temperature.  But  this  is  not  all.  If  frogs,  after  being 
injected  with  tetanus,  are  subjected  to  a  high  temperature  for  one 
day,  and  then  placed  in  the  refrigerator,  they  will  not  become  sick. 
But  on  bringing  the  animals  back  into  higher  temperatures  after 

1  Arch.  Internat.  de  Pharmacodynam.,  Vol.  7,  1900. 


TOXIN  AND  ANTITOXIN.  537 

the  lapse  of  weeks  or  months,  it  will  be  found  that  they  sicken  after 
a  shortened  period  of  incubation.  Are  any  further  proofs  of  the 
slow  action  of  the  toxophore  group  required? 

It  is  not  easy  to  meet  all  of  Gruber's  statements  because  he  fre- 
quently makes  use  of  misleading  tactics.  He  often  reaches  the 
same  conclusions  as  1  myself,  and  grants  that  certain  of  my  views 
are  permissible  or  probable.  In  some  things,  he  says,  I  am  correct 
in  the  main,  in  others  I  may  be  right,  but  have  not  strictly  proved 
my  point.  All  these  statements  are  but  a  clever  contrivance  to 
give  the  reader  the  impression  that  my  theory  is  but  a  product  of 
the  imagination  when  as  a  matter  of  fact  is  it  really  a  hypothesis 
developed  experimentally.  This  brings  me  to  Gruber's  fifth  con- 
clusion. 

5.  "  The  development  of  antitoxin  has  no  connection  whatever 
with  toxic  action  or  cell  immunity." 

It  will  suffice  for  me  to  call  attention  to  the  fact  that  I  have  always 
insisted  on  distinguishing  between  the  haptophore  and  toxophore 
groups  in  the  toxin  molecule  and  also  between  the  anchoring  and  the 
action  of  poison.  I  might  add  that  this  absolute  independence  of 
toxic  action  and  antibody  production  is  a  principle  which  1  formu- 
lated, not  Gruber.  As  far  back  as  1898,  Weigert l  rightly  pointed 
out  that  my  demonstration2  of  antitoxin  production  through  non- 
poisonous  toxoids  was  sufficient  to  demonstrate  the  independence 
of  antitoxin  production  and  toxic  action.  Furthermore  I  have 
repeatedly  pointed  out  that  the  development  of  antitoxin  depends 
on  the  haptophore  group.  Over  1£  years  ago  Paltauf3  called 
Gruber's  attention  to  the  weak  points  in  his  objection  and  one  might 
therefore  have  expected  that  Gruber  would  not  again  bring  forward 
this  old  fairy-tale.  In  the  future  I  shall  not  reply  to  perversions 
of  this  kind. 

So  far  as  the  reasons  are  concerned,  which  Gruber  gives  in  sup- 
port of  the  above  statement  regarding  the  development  of  anti- 
toxin, I  may  at  once  say  that  I  can  assent  to  them  word  for  word- 
Thus  the  statement  that: 

(a)  "  Many  substances  which  are  entirely  innocuous  lead  to 
the  formation  of  antibodies''  is  the  first  consequence  of  my  viewy 
and  experimental  labors.  The  fact  that 

1  Lubarsch-Ostertag,  Ergebnisse  der  pathologischen  Anatomie,  IV  Jahrgang. 

2  Werthbemessung  des  Diphtherieheiiserums,  Klin.  Jahrbuch. 

3  Wiener  klin.  Wochenschr.  No.  49,  1901. 


538  COLLECTED  STUDIES   IN    IMMUNITY. 

(6)  "  Certain  animals  non-susceptible  to  certain  toxins  never- 
theless produce  antibodies  "  needs  no  further  explanation  according 
to  my  theory.  Certain  species  of  animals  may  possess  suitable 
receptors  for  binding  the  toxin  and  producing  antitoxin  although 
their  cells  are  insensitive  to  the  action  of  the  toxophore  group.  Accord- 
ing to  Metchnikoff  this  seems  often  to  be  the  case  with  tetanus  toxin 
in  crocodiles.  As  already  pointed  out  years  ago  by  Weigert l  accord- 
ing to  my  theory,  the  production  of  antitoxin  need  not  at  all  be 
preceded  by  any  injury  in  a  clinical  sense.  In  fact,  too  strong  an 
injury  may  cause  the  cell  to  lose  its  power  of  regeneration,  owing 
to  the  toxic  action  on  the  vital  group  [Leistungskern],  For  example, 
if  a  specific  nerve  poison  is  anchored  by  a  fitting  receptor  of  an  indiffer- 
ent cell  (liver)  we  should  expect  the  production  of  an  antibody  by 
the  liver,  even  if  the  liver-cell  does  not  become  tetanized.  In  my 
address  at  Hamburg2  before  the  Congress  of  Naturalists-  I  pointed 
out  that  the  local  origin  of  antitoxin,  which  Romer  deduces  from 
his  splendid  experiments  with  abrin,  will  often  make  it  possible  to 
transfer  part  of  the  antitoxin  production  from  the  vital  organs  to 
the  indifferent  connective  tissue,  by  means  of  subcutaneous  injec- 
tion of  poison. 

Gruber's  next  statement  is: 

(c)  "  Despite  a  plentiful  production  of  antibody,  the  suscep- 
tibility to  the  poison  may  remain,  or  even  increase." 

I  have  already  discussed  the  principle  of  hypersensitiveness 
.and  mentioned  the  fact  that  this  objection  restrained  me  for  a  long 
time  from  publishing  my  theory.  But  even  these  phenomena  were 
satisfactorily  explained  in  accordance  with  the  side-chain  theory, 
by  the  assumption  of  an  increase  of  affinity  and  a  rupture  of  the 
toxin-antitoxin  combination.  To  be  sure  it  is  possible  that  our 
explanation  touches  but  part  of  the  subject,  and  that  in  reality  the 
phenomena  are  far  more  complex.  But  this  is  no  reason  for  seek- 
ing to  overthrow  the  theory;  to  do  so  would  be  to  completely  mis- 
apprehend the  purpose  of  a  theory.  Surely  one  cannot  demand 
that  a  theory  will  at  once  explain  all  the  complex  phenomena  of 
so  difficult  a  subject  as  this.  A  theory  ought  primarily  to  possess 
heuristic  value,  pointing  out  new  paths  into  a  complex  subject;  it 
should  smooth  the  way.  The  actual  research  must  be  left  to  the 
scientific  investigator.  Science  can  be  advanced  only  by  means 

1  1.  c.  l  Deutsche  med.  Wochenschr.  1901. 


TOXIN  AND  ANTITOXIN.  539 

of  experimental  analysis,  and  not  by  high-flown  words  of  a  mis- 
leading dialectic. 

(d)  "Cell  immunity  can  be  acquired  without  the  formation  of 
antibodies." 

This  statement,  too,  does  not  surprise  me.  All  that  the  side- 
chain  theory  aims  to  do  is  to  explain  how  the  production  of  anti- 
bodies may  be  conceived.  But  I  have  never  yet  claimed  that  this 
is  the  only  means  by  which  the  organism  can  defend  itself  against 
deleterious  influences.  I  would  call  attention  particularly  to  the 
vSixth  Communication  on  Hsemolysins,1  in  which  Morgenroth  and  I 
pointed  out  that  not  all  substances  capable  of  being  anchored  need 
necessarily  excite  the  production  of  antibodies.  We  have  always 
emphasized,  however,  that  immunity  may  be  developed  despite 
this,  chiefly  through  a  disappearance  of  receptors.2  In  our  isolysin 
experiments  we  observed  that  the  blood-cells  became  insusceptible 
and  we  demonstrated  that  this  was  due  to  a  lack  of  receptors.  The 
interesting  fact  observed  by  Kossel  and  by  Camus  and  Gley  that 
during  the  course  of  immunization  with  eel  blood,  the  blood-cells 
of  rabbits  acquire  a  high  resistance  against  that  poison,  is  probably 
most  easily  explained  by  assuming  that  the  cells  acquired  immunity 
in  the  way  above  mentioned. 

This,  of  course,  does  not  exhaust  the  possibilities  of  the  origin 
of  immunity  not  due  to  antitoxins.  Thus  under  the  influence  of 
the  anchored  poison  new  receptors  may  be  formed  which  are  so  firmly 
united  to  the  protoplasm  that  they  are  not  thrust  off.  Such  receptors 
Morgenroth  and  I  have  therefore  termed  "sessile  receptors."  If 
the  production  of  such  an  excess  of  receptors  takes  place  in  a  rather 
indifferent  tissue,  as  in  connective  tissue,  it  will  readily  be  seen  how 
the  receptors  can  serve  to  deflect  the  poison,  and  produce  a  more 
or  less  marked  immunity.  In  that  case  on  comparing  a  normal 
animal  with  an  immunized  one,  the  conditions  would  be  like  those 
observed  with  tetanus  poison  in  normal  guinea-pigs  and  normal 
rabbits,  respectively.  The  studies  of  Donitz  and  Roux  have  shown 
that  the  guinea-pig  possesses  receptors  for  tetanus  toxin  only  in 
the  brain,  whereas,  rabbits,  in  addition  to  the  receptors  in  the  cen- 
tral nervous  system,  possess  about  thirty  times  as  many  such  recep- 
tors outside  this  system. 

1  See  page  88. 

2Schlussbetrachtungen  in  Xothnagel's  Handbuch.,  Vol.  VIII. 


540  COLLECTED  STUDIES  IN   IMMUNITY. 

Another  possibility  of  cell  immunity  is  that  the  protoplasm  of 
cells  which  are  ordinarily  susceptible  is  no  longer  affected  by  cer- 
tain poisons.  This  kind  of  immunity,  which  to  be  sure  1  consider 
very  rare,  would  correspond  to  mithridatism  or  acquired  tolerance 
in  the  old  sense.  A  fourth  possibility,  finally,  is  the  adaptation  of 
the  phagocytic  apparatus  in  MetchnikofPs  sense. 

It  is  obvious,  of  course,  that  all  the  sevarious  subordinate  kinds 
of  immunity  occur  alone  as  well  as  in  manifold  combinations.  Thus, 
as  already  mentioned,  immunization  with  eel  blood  is  followed  by 
antitoxin  immunity  and  tissue  immunity.  In  the  lower  animals, 
however,  which  as  Metchnikoff  has  shown  are  but  little  adapted 
to  the  production  of  antitoxin,  other  defensive  contrivances  leading 
to  cell  immunity  will  predominate  From  this  point  of  view  there- 
fore the  condition  described  by  Gruber,  namely,  that  frogs  can  be 
immunized  against  abrin  without  their  showing  any  antitoxin,  offers 
no  difficulty.  So  far  as  the  frog  is  concerned  the  only  question  is 
which  kind  of  cell  immunity  is  present,  i.e.,  whether  there  is  a  dis- 
appearance of  receptors,  or  whether  there  are  sessile  receptors,  etc.1 

In  view  of  the  detailed  statements  given  above  I  presume  I  need 
add  nothing  to  the  following  passage  in  Gruber's  conclusion : 

(e)  "The  production  of  antibodies  takes  place  at  entirely  different 
localities  than  does  toxic  action." 

The  discerning  reader  will  at  once  see  that  this  statement  does 
not  in  the  least  contradict  my  views.  In  fact  it  is  merely  another 
way  of  expressing  what  is  really  the  nucleus  of  my  theory.  The 
generalization,  however,  is  false,  that  the  production  of  antibody 
necessarily  takes  place  in  localities  different  from  those  in  which 
toxic  action  occurs.  If  Gruber  therefore  believes  that  this  riddles 
my  theory  it  is  evident  that  he  understands  the  principles  under- 

1  Gruber  cites,  as  a  serious  objection  to  my  theory,  that  Madsen  observed 
immunity  in  a  rabbit  which  had  been  immunized  with  diphtheria  toxin,  and 
yet  was  unable  to  find  antitoxin  in  the  blood.  I  will  only  say  that  Madsen 
did  not  find  the  blood  entirely  free  from  antitoxin  since  he  tested  the  serum 
only  to  1/10  I.  E.  Small  quantities  of  antitoxin  could  be  very  well  have  been 
present  and  these,  of  course,  would  be  of  considerable  importance  for  the  ques- 
tion as  to  whether  this  was  a  case  of  entire  absence  of  antitoxin.  Besides 
this  I  may  add  that  in  diphtheria  poison  the  case  reported  by  Madsen  must 
be  extremely  rare.  During  the  course  of  many  years  the  different  Serum 
Institutes  have  immunized  thousands  of  different  animals  against  diphtheria. 
In  all  this  time,  however,  I  have  never  learned  of  a  case  analogous  to  Madsen's, 
either  from  the  literature  or  from  private  sources. 


TOXIN   AND   ANTITOXIN  541 

lying  my  views  no  better  than  he  did  two  years  ago.  At  that  time 
Paltauf 1  tried  in  vain  to  make  this  elementary  consequence  of  the 
side-chain  theory  comprehensible  to  him. 

Gruber's  sixth  conclusion  is  as  follows: 

6.  "  The  specific  antibodies  are  not  normal  body  constituents. 
They  are  newly  formed  only  after  the  introduction  of  foreign  sub- 
stances. This  new  formation  has  the  character  of  an  internal  secre- 
tion." 

So  far  as  the  first  point  is  concerned  one  cannot  help  being  amazed 
at  the  lack  of  literary  knowledge  which  permits  an  author  to  make 
such  statements.  1  need  only  refer  to  the  studies  of  Pfeiffer,  Bordet, 
Flexner,  Kraus,  Bail,  Peterssen,  etc.,  or  to  the  comprehensive  resume 
by  M.  Neisser 2  concerning  the  antibodies  found  in  normal  serum, 
The  literature  on  normal  antibodies  of  various  kinds  is  very  large, 
and  yet  has  been  entirely  ignored  by  Gruber.  Thus  amboceptors 
against  different  bacteria  (cholera,  typhoid,  anthrax),  antiambo- 
ceptors,  anticomplements,  antitoxins,  antiterments,  etc.,  have  been 
observed.  I  shall,  however,  mention  merely  a  few  points  which 
may  be  of  special  interest. 

i.  The  very  frequent  occurrence  of  diphtheria  antitoxin  in  horses 
(Meade,  Roux,  Bolton,  Cobbett).  In  view  of  the  high  percentage 
of  this  occurrence,  the  attempts  to  ascribe  this  antitoxin  in  normal 
horse  serum  to  a  diphtheria  running  a  latent  course  must  be  regarded 
as  failures.  Since  this  phenomenon  has  been  observed  in  about 
30%  of  the  horses,  it  is  surely  not  reasonable  to  assume  that  an 
occurrence  of  diphtheria  in  horses  should  so  frequently  have  entirely 
escaped  the  large  number  of  excellent  observers  representing  animal 
pathology.  Such  a  frequency  of  the  disease  should,  of  course,  also 
have  manifested  itself  epidemiologically.  The  fact  that  in  one  single 
instance  Cobbett  observed  a  diphtheritic  infection  in  a  horse  cer- 
tainly does  not  alter  the  circumstances. 

ii.  I  must  mention  the  interesting  observations  made  by  v.  Dun- 
gern  3  that  normal  rabbit  serum  contains  an  antibody  against  that 
substance  in  star-fish  eggs  which  is  toxic  for  sea-urchin  spermatozoa. 
I  am  sure  that  no  one,  just  to  please  Gruber,  will  assume  that  there 
is  any  connection  between  rabbits  and  star-fish  and  their  eggs 

in.  Laveran  has  found  that  the  blood  of  healthy  human  beings 

1  Wiener  klm.  Wochenscbr.  1901,  No.  49. 

2  Deutsche  med.  Wochenschr.  1900. 

3  Zeitschr.  f.  allgemeine  Physiologic,  Vol.  1,   1901. 


542  COLLECTED  STUDIES  IN   IMMUNITY. 

contains  a  substance  which  kills  trypanosomes,  whereas  this  is  not 
present  in  the  blood  of  other  animals  and  cannot  be  produced  in  so 
large  an  amount  even  by  immunization.  This  might  be  the  reason 
why  (aside  from  sleeping  sickness  of  Central  Africa)  man  is  so  refrac- 
tory toward  trypanosome  infection. 

But  if  such  a  wealth  of  facts  is  disregarded  in  statements  con 
cerning  "  our  certain  knowledge,"  it  must  be  admitted  that  a  scientific 
discussion  is  entirely  out  of  the  question,  and  had  best  be  avoided 
in  the  future. 

Furthermore,  so  far  as  conceiving  the  production  of  antitoxin 
to  be  a  secretion  is  concerned,  I  may  say  that  this  part  of  the  paper 
is  nothing  but  another  way  of  stating  what  I  have  always  held. 
Paltauf,1  for  instance,  pointed  this  out  to  Gruber  some  years  ago, 
"  In  passing  I  may  say  that  an  '  escape  '  of  particles  of  protoplasm 
into  the  blood  really  denotes  a  secretion.''  In  an  address  delivered 
in  1899  (!)  I  expressed  myself  in  a  way  that  shows  that  I  have 
always  considered  the  production  of  antitoxin  to  be  a  secretory 
process.2 

"  Or,  s'il  y  a  lieu  de  croire  que  les  Antitoxines  doivent  leur  origine 
a  une  sorte  de  fonction  secretaire  des  cellules  et  ae  sont  par  conse- 
quent nullement  etrangeres  a  1'organisme,  le  rapport  specinque  qui 
les  unit  avec  leurs  toxines  n'en  devient  que  plus  etrange." 

This  point  has  been  demonstrated  especially  by  the  researches 
of  Salomonsen  and  Madsen,  and  of  Roux  and  Vaillard. 

But  just  this  secretory  character  of  antibody  production  is  abso- 
lutely at  variance  with  the  older  view  that  antitoxins  are  merely 
transformation  products  of  the  toxins.  This  was  the  view  defended 
by  Buchner  and  held  to  be  possible  by  Gruber  even  in  his  last  attack. 
It  is  just  as  impossible  to  believe  that  antitoxins  arise  from  toxins 
as  it  is  to  believe  that  lipase  is  transformed  fat,  or  amylase,  trans- 
formed starch. 

Thus  we  see  that  the  various  points  brought  up  by  Gruber  are 
nothing  but  reproductions  of  my  views;  the  little  that  deviates  is 
incorrect  or  is  based  on  misconceptions  of  an  inflated  knowledge 
of  the  literature. 

Grubers  last  two  conclusions  contain  so  little  that  is  new  that 
it  hardly  pays  to  discuss  them.  For  completeness'  sake,  however. 
I  shall  append  them. 

1  Weiner  klin.  Wochenschr.  1901,  No  49. 

'  This  appeared  only  as  an  abstract  in  La  Semaine  Medicale,  1899. 


TOXIX  AND  ANTITOXIN.  543 

7.  "The  power  to  excite  the  formation  of  antibodies  is  due  to 
certain  peculiarities  in  the  chemical  structure  of  the  substance  which 
excites  this  antibody  production.     A  prerequisite  for  this  produc- 
tion as  well  as  for  toxic  action  is  the  chemical  union  of  the  foreign 
substance  with  certain  particular  constituents  of  the  cells." 

This,  I  may  say,  is  a  short,  though  not  particularly  good,  resume 
of  the  side-chain  theory. 

8.  "The   non-poisonous   toxin-antitoxin   combination   also   lacks 
the  power  to  excite  the  production  of  antitoxin.     The  entire  chemi- 
cal character  of  this  combination  is  different  from  that  of  the  uncom- 
bined  substances." 

This,  too,  is  one  of  the  fundamental  principles  of  my  theory,  and 
is  most  readily  explained  by  the  assumption  that  the  antitoxin  fits 
into  the  same  group  which  effects  the  union  of  the  toxin  with  the 
susceptible  cells.  Furthermore,  I  really  see  no  reason  why  Gruber 
should  make  a  special  point  of  the  fact  that  the  chemical  character 
of  the  toxin-antitoxin  combination  has  changed.  That  is  merely 
a  trick  of  speech  which  will  make  but  little  impression  on  the  scientific 
reader. 

That  the  antitoxins  are  nothing  but  thrust-off  receptors  capable 
of  uniting  with  the  poison — this  assumption,  together  with  its 
immediate  consequence  that  the  toxin-antitoxin  combination  must 
be  non-poisonous,  is  the  key  to  my  entire  theory.  We  are,  in 
fact,  dealing  with  an  extremely  important  law  which  Weigert 
and  I  compared  to  the  principle  of  the  lightning-rod  and  which 
v.  Behring  briefly  expressed  as  follows:  "The  same  substance  in 
the  living  body  which,  when  in  the  cell,  is  the  prerequisite  of  a  poison- 
ing, becomes  the  healing  agent  when  it  is  present  in  the  blood.'7 
This  law  applies  not  only  to  the  toxins  but  possesses  general  applic- 
ability. I  may  here  refer  the  reader  to  Ransom's  experiments,  which 
show  that  the  cholesterin  in  the  red  blood-cells  causes  haemolysis 
by  saponin,  while  at  the  same  time  the  cholesterin  of  the  serum 
causes  an  inhibition  of  this  poisoning. 

Gruber,  however,  thinks  that  it  has  not  been  proved  that  the 
haptophore  group,  which  anchors  the  toxin  to  the  vital  constituent 
of  the  protoplasm,  is  the  same  which  anchors  the  toxin  to  the  anti- 
toxin. A  year  and  a  half  ago  he  expressed  this  quite  clearly  as 
follows : l 

"Ehrlich  may  have  demonstrated  that  the  toxin  is  bound  to 
1  Wiener  klin.  Wochenschrift,  1901,  No.  50. 


544  COLLECTED   STUDIES  IN   IMMUNITY. 

the  antitoxin  by  a  combining  group  which  differs  from  the  toxo- 
phore  group.  But  where  and  how  has  he  shown  that  the  toxin  in 
addition  to  its  toxophore  group  possesses  only  one  haptophore  group, 
namely,  the  one  which  combines  with  the  antitoxin?  How  has  he 
shown  that  the  same  haptophore  group  acts  in  all  chemical  reac- 
tions of  the  toxin?  On  the  contrary  it  can  positively  be  stated  that 
the  toxin  must  necessarily  be  a  very  complex  molecule  possessing 
many  different  haptophore  groups.  Here,  gentlemen,  lies  the  root 
of  the  evil.  All  this  misconception  of  the  side-chain  theory  would 
have  been  impossible  but  for  the  mistake  in  the  choice  of  an  article; 
i.e.,  if  Ehrlich  instead  of  speaking  of  the  haptophore  group  had  said 
a  haptophore  group." 

So  this  is  my  great  fault,  the  choice  of  an  article!  I  may  leave 
it  for  the  reader  to  decide  how  weighty  this  objection  is.  Never- 
theless let  us  see  what  Gruber  really  means. 

Let  us  assume,  for  example,  that  a  poison,  in  addition  to  the 
toxophore  group,  possesses  two  different  groups  with  haptophore 
functions.  One  of  these,  group  a,  corresponds  to  what  my  theory 
demands,  since  it  is  able  to  combine  with  a  receptor  of  the  cell.  As 
a  result  of  this  combination,  however,  there  is  to  be  not  an  over- 
production of  a  receptor  fitted  to  a,  but  the  production  of  a  differ- 
ent substance,  fitting  the  second  haptophore  group,  6,  of  the  toxin. 
It  will  at  once  be  seen  that  this  entire  premise  of  Gruber  is 
very  artificial  and  unnatural.  One  can  easily  understand  that  the 
blocking  of  a  given  group  can  cause  a  new  development  of  the  same 
group.  This  corresponds  to  Weigert's  fundamental  law  of  regenera- 
tion. But  it  is  very  difficult  to  comprehend  how  the  blocking  of 
one  group,  a,  would  always  lead  to  the  development  of  a  different 
group,  6.  Furthermore,  it  is  incomprehensible  why  at  least  part 
of  the  poison  by  means  of  its  haptophore  group  b  should  not  be 
anchored  by  a  combining  substance  preformed  in  the  cell,  a  substance 
which  can  therefore  act  as  a  receptor.  If  the  toxin  really  possessed 
two  haptophore  groups,  a  and  6,  it  would  be  possible  and  probable 
that  two  different  antitoxins  would  be  developed  by  the  cell.  But  that 
is  a  question  easily  decided  experimentally,  and  one  which  has  been 
studied  in  this  Institute  for  years.  During  all  this  time  we  have 
never  discovered  even  the  slightest  reason  for  believing  that  diph- 
theria serum,  obtained  from  different  animals  and  by  means  of  differ- 
ent cultures,  possesses  any  such  complex  constitution  as  Gruber's 
view  would  require. 


TOXIN   AND   ANTITOXIN  545 

We  see,  therefore,  that  the  first  step  taken  with  the  aid  of  Gruber's 
hypothesis  leads  us  astray.  But  when  we  attempt  to  see  how  the 
antitoxin  could  act  according  to  Gruber's  scheme,  we  find  ourselves 
lost  in  a  maze.  The  antibody  secreted  by  the  cell  is  to  combine 
with  a  collateral  group  6,  of  the  toxin,  leaving  group  a,  which  pri- 
marily effected  the  anchoring  of  the  poison,  intact.  How  then  is 
any  antitoxic  effect  to  take  place?  One  might  perhaps  assume  that 
by  the  occupation  of  group  b,  the  toxin  loses  its  toxicity  through 
some  influence  exerted  on  the  toxophore  group.  The  poison  would 
thus  in  a  certain  sense  be  changed  into  a  toxoid  by  the  occupation 
of  group  b.  In  that  case,  however,  the  toxin  with  group  b  neutralized, 
should  still  be  able  to  excite  the  production  of  antitoxin,  just  as  toxoids 
do.  As  a  matter  of  fact,  this  is  not  at  all  the  case,  for  we  know  that 
toxin  neutralized  with  antitoxin  has  completely  lost  both  its  toxic 
property  and  its  power  to  produce  antitoxin.  This  fact,  which  is 
absolutely  irreconcilable  with  a  plurality  of  the  haptophore  groups, 
is  easily  explained  by  my  theory  by  a  blocking  of  the  haptophore 
group  of  the  toxin. 

We  see,  therefore,  that  Gruber's  assumption  leads  to  consequences 
which  are  absolutely  untenable.  It  certainly  is  far  from  being  an 
improvement  on  my  theory.  In  general,  also,  the  principles  of 
scientific  investigation  demand  that  we  restrict  ourselves  to  the 
simplest  explanations  possible  and  only  make  use  of  more  complex 
ones  if  it  is  absolutely  necessary.  But  there  is  not  the  least  reason 
for  Gruber's  assumption  of  several  haptophore  groups;  on  the  con- 
trary there  are  a  large  number  of  objections  to  it. 

By  this  I  do  not  mean  to  say  that  in  addition  to  the  haptophore 
and  toxophore  group  the  toxin  molecule  contains  no  other  chemical 
groups,  such  as  amido  or  aldehyde  groups,  which  are  able  to  com- 
bine with  other  bodies.  I  merely  contend  that  these  atomic  groups 
do  not  influence  the  specific  immunizing  process. 

To  take  a  chemical  example,  it  is  possible  by  diazotizing  all  kinds 
of  amins  to  transform  these  into  diazo  combinations  which,  corre- 
sponding to  the  original  substance  employed,  contain  other  radicals 
capable  of  reacting,  thus  COH,  CN,  OH,  NO,  etc.  The  specific  prop- 
erty of  these  substances,  that  is,  the  property  of  forming  azo  dyes, 
is,  however,  connected  exclusively  with  the  N-N  group.  The  reac- 
tions which  the  other  groups  can  enter  into  have  nothing  to  do  with 
this  specific  reaction.  I  conceive  the  constitution  of  the  toxins 
to  be  similar  in  character. 


546  COLLECTED  STUDIES  IN  IMMUNITY. 

A  few  words,  now,  concerning  the  side-chain  theory  and  immunity 
Gruber  himself  has  found  that  this  theory  is  constantly  gaming 
ground,  while  I  am  gratified  to  see  it  treated  in  detail  in  the  best 
text-books  as  well  as  in  excellent  digests  compiled  by  a  large  num- 
ber of  my  colleagues.1  In  addition  to  this  hundreds  of  separate 
studies  have  been  based  on  the  side-chain  theory  so  that  I  may  well 
believe  that  it  best  serves  to  explain  the  facts  already  observed  as 
well  as  to  allow  new  facts  to  be  predicted.  Gruber's  appeal,2  there- 
fore, that  "Ehrlich's  theory  is  a  great  mistake,  and  is  bound  soon  to 
disappear  from  the  scientific  arena,"  has  had  but  little  success;  in 
fact  it  seems  to  have  had  the  contrary  effect.  The  large  number 
of  investigators,  who  are  constantly  eagerly  working  on  the  prob- 
lems of  immunity  know  what  is  best  for  them,  and  will  not  be  dictated 
to  against  their  own  experience  and  conviction  by  one  who  seeks 
to  make  up  his  own  lack  of  experimental  work  in  this  complex  domain, 
by  superficial  studies  of  the  literature.  Gruber,  for  instance,  says 
that  his  original  failure  was  due  perhaps  to  the  fact  "  that  a  few 
of  his  experiments  proved  not  to  be  quite  sufficient."  This  is  a 
mild  expression  in  view  of  the  fact  that  every  one  of  Gruber's  experi- 
ments directed  against  my  views  has  been  shown  to  be  fallacious. 
The  studies  in  which  his  errors  were  pointed  out  and  demonstrated 
experimentally  have  all  been  published  in  detail.3  The  result,  as 
usual,  was,  that  after  the  corrections  had  been  made,  Gruber's  attacks 
proved  to  be  additional  supports  for  my  theory.  Gruber  has  not 
replied  to  these  articles,  despite  the  long  time  since  their  publication. 
Perhaps  he  thinks  the  less  said  the  better. 

I  have  finished.  I  must  almost  wonder  why  this  detailed  reply 
to  an  attack  whose  virulence  and  unusual  tone  are  almost  a  con- 
firmation of  my  views.  But  I  have  thought  it  my  duty  to  guide  the 
reader  through  the  intricate  maze  of  Gruber's  statements  because  I 
feel  that,  owing  to  the  large  number  of  misconceptions  and  mislead- 
ing arguments  which  they  contain,  a  field  of  investigation  full  of 
promise  might  become  discredited. 

1  I  may  mention  those  of  Aschoff,  v.  Dungern,  Grunbaum,  Levaditi,  Sachs 
Tavel,  Wassermann,  Welch,  Bruck. 

3  Wiener  klin.  Wochensch.  1901,  No.  44. 

8  Sachs,  Berl.  klin.  Wochensch.  1902,  Nos.  9  and  10;  Ehrlich  und  Sachs, 
same  journal,  1902,  No.  21;  Morgenroth  and  Sachs,  same  journal,  1902,  Nos. 
27  and  35;  Marx,  Zeitsch.  f.  Hyg.,  Bd.  40,  1902;  Wechsberg,  Wiener  klin. 
Wochensch.  1902,  Nos.  13  and  28. 


XXXIX.  THE  RELATIONS  EXISTING  BETWEEN  TOXIN 
AND    ANTITOXIN   AND  THE  METHODS   OF  THEIR 
STUDY.1 

BY 

Prof.  PAUL  EHRLICH  and  DR.  HANS  SACHS. 

THE  subject  of  toxins  and  antitoxins,  although  representing  one 
of  the  best  studied  domains  of  biology,  is  still  the  subject  of  lively 
controversy.  The  difficulties  which  beset  exact  studies  are  obvious. 
We  are  dealing  with  substances  which,  for  the  present  at  least,  are 
of  unknown  chemical  constitution  and  which  we  are  compelled  to 
employ  in  the  form  presented  by  the  life  activities  of  vegetable  or 
animal  organisms,  i.e.,  in  an  impure  state  and  mixed  with  countless 
other  products  of  the  living  body.  All  attempts  to  isolate  these 
bodies  and  discover  their  chemical  character  encounter  endless  diffi- 
culties, so  that,  if  we  consider  their  great  significance  in  practical  medi- 
cine, it  almost  seems  ironical  for  nature  to  offer  these  substances  to 
man  in  such  an  unstable  and  variable  form.  In  spite  of  this,  however, 
scientific  investigations  have  been  able  to  obtain  a  deep  insight  into 
the  nature  and  mode  of  action  of  toxins  and  antitoxins;  and  since 
chemical  means  could  not  be  employed,  it  remained  for  the  experi- 
mental biologist  to  undertake  these  studies.  In  place  ot  chemical 
analysis,  therefore,  we  have  the  biological  reaction,  which  in  the  case 
of  toxins  is  the  characteristic  toxic  action,  in  the  case  of  antitoxins 
the  property  of  specifically  influencing  or  inhibiting  this  action. 

An  event  of  considerable  importance  was  the  introduction  of  the 
quantitative  method  of  study  by  Ehrlich,  a  method  which  opened 
the  way  for  the  present  development  of  immunity  studies.  At  the 
same  time  Ehrlich's  introduction  of  test-tube  experiments  (haemag- 
glutination,  haemolysis),  by  avoiding  the  individual  fluctuations  of 

1  Uber  die  Beziehungen  zwischen  Toxin  und  Antitoxin  und  die  Wege  ihrer 
Erforschung,  Leipzig,  1905,  Gustav  Fock. 

547 


548  COLLECTED  STUDIES  IN   IMMUNITY 

animal  experiments,  furnished  a  more  exact  basis,  so  that  the  mathe- 
matical harmony  of  toxin-antitoxin  experiments  in  vivo  and  in  vitro 
became  very  convincing.  At  the  present  time,  therefore,  we  may 
regard  it  as  almost  axiomatic  that  toxin  and  antitoxin  act  on  each 
other  chemically  and  without  the  intervention  ot  vital  forces. 

These  quantitative  biological  studies,  however,  have  not  merely 
thrown  light  on  the  relations  existing  between  toxin  and  antitoxin t 
but  have  also  given  us  valuable  intormation  concerning  the  constitu- 
tion of  the  poisons  themselves.  Almost  at  the  outset  it  was  found 
that  the  two  properties  of  toxins  which  could  be  analyzed,  namely, 
poisonous  action  and  the  property  to  bind  antitoxin,  do  not  at  all 
go  hand  in  hand.  In  this  connection  the  continuous  study  of  toxin 
solutions  which  are  allowed  to  stand  for  some  time  proved  particu- 
larly instructive,  for  it  was  found  that  while  the  power  to  bind  anti- 
toxin remained  constant,  the  toxicity  gradually  dminished.  This 
study  gave  us  one  of  the  fundamental  conceptions  underlying  the 
modern  view  of  toxins,  namely,  that  toxicity  and  combining  power 
are  two  distinct  and  independent  properties  of  the  toxin  molecule. 
As  is  well  known,  this  fact  is  expressed  by  the  side-chain  theory  by 
assuming  that  the  toxin  molecule  possesses  two  specific  atomic  groups, 
one  of  which  is  toxophore,  the  other  haptophore.  Destruction  or 
loss  of  the  toxophore  group  gives  rise  to  the  non-toxic  toxoids  which 
are  still  capable  of  binding  antitoxin.  As  a  result  of  the  high  degree 
of  lability  of  the  toxophore  group,  this  transformation  into  toxoid  is 
a  spontaneous  process.  And  since  the  production  of  effective  bacterial 
toxin  solutions  takes  a  certain  time,  it  is  obvious  that  we  can  practi- 
cally never  obtain  a  pure  toxin  consisting  entirely  of  similar  molecules. 
All  our  work  must  be  done  with  toxic  solutions  which,  even  if  we 
assume  that  the  bacteria  have  produced  only  a  single  primary  toxin, 
represent  a  "mixture  of  toxin  and  toxoid. 

But  do  the  bacilli  secrete  only  a  single,  homogeneous  poison? 
This  question  has  come  more  and  more  to  be  the  subject  of  an  ani- 
mated discussion.  Closely  associated  with  it  is  the  further  question 
as  to  the  nature  of  the  reaction  which  occurs  when  toxin  and  anti- 
toxin unite.  The  study  of  these  problems  was  made  possible  by 
an  important  extension  of  quantitative  toxin  analysis,  namely, 
Ehrlich's  method  of  partial  neutralization.  This  consists  essentially 
in  mixing  a  constant  amount  ot  poison  with  varying  amounts  of  anti- 
toxin and  then  determining  the  toxicity  of  the  various  mixtures, 
i.e.,  the  decrease  in  toxicity  brought  about  by  each  successive  addi- 


TOXIN   AND   ANTITOXIN:    METHODS  OF  THEIR   STUDY      549 

tion  of  antitoxin.  By  means  of  a  graphic  representation  ot  the 
figures  thus  obtained,  we  can  get  a  deeper  insight  into  the  details 
of  the  combining  phenomena.  Even  now,  after  physical  chemistry 
has  taken  such  great  interest  in  the  reactions  between  toxin  and 
antitoxin,  all  the  various  statements  concerning  the  subject  are 
finally  based  on  the  method  of  partial  neutralization. 

From  the  outset  Ehrlich  felt  sure  that  toxin  and  antitoxin  could 
not  be  simple  substances  of  strong  affinities  which  combined,  for 
instance,  like  caustic  soda  and  hydrochloric  acid.  This  was  evi- 
denced particularly  by  the  phenomenon  which  has  often  been  termed 
the  "inequality"  of  serum  experiments.  Thus  if  varying  amounts 
of  toxin  are  added  to  a  constant  amount  of  antitoxin  (an  immune 
unit),  two  distinct  limits  will  be  obtained:  L0  (  =  Limit  zero)  is  the 
quantity  of  toxin  in  which  the  mixture  is  just  completely  non-toxic, 
i.e.,  physiologically  neutral.  Lf  (  =  Limit  death)  is  the  quantity  of 
toxin  in  which  the  mixture  is  still  just  able  to  exert  all  its  character- 
istic toxin  action,  i.e.,  in  the  case  of  diphtheria  poison  to  just  kill 
the  guinea-pig  acutely.  Now  if  toxin  and  antitoxin  behaved  like 
caustic  soda  and  hydrochloric  acid,  the  difference  between  Lf  and  L0, 
which  we  shall  term  D,  should  correspond  to  one  lethal  dose  (L  D.) 
As  a  matter  of  fact,  however,  D  is  usually  considerably  larger,  so  that 
our  first  inequality  becomes  Lf  — Lo>L.  D. 

Hence  only  two  possibilities  exist.  Either  toxin  and  antitoxin 
react  with  one  another  like  a  weak  base  and  a  weak  acid  (e.g.,  am- 
monia and  boric  acid),  in  which  case  the  high  value  of  D  is  the  expres- 
sion of  an  incomplete  neutralization,  or  else  the  poison  solution, 
besides  the  real  toxin,  contains  a  second  substance  of  less  affinity. 
This  substance,  while  unable  to  produce  the  characteristic  toxin 
effects,  gives  rise  to  certain  mild  toxic  phenomena.  In  the  case  of 
diphtheria  poison  (owing  to  the  practical  importance  ot  diphtheria 
antitoxin,  the  discussion  has  usually  centered  around  this  poison) 
human  pathology  had  long  taught  that  acute  diphtheria  infection 
is  often  followed  by  a  second  set  of  intoxication  phenomena,  namely, 
the  peculiar  paralyses  which  develop  after  the  acute  disease  has  dis- 
appeared. A  priori,  therefore,  the  assumption  was  highly  probable 
that  the  high  value  of  D  was  due  to  different  components  of  the  poison. 
And  when  the  results  of  clinical  experience  and  animal  experiments 
harmonized  so  perfectly,  the  probability  became  almost  a  certainty. 
It  has  been  found  that  the  toxicity  of  mixtures  whose  toxin  content 
lies  between  L0  and  Lt  is  not  quantitatively  diminished,  but  is  actually 


550  COLLECTED  STUDIES  IN  IMMUNITY. 

different  qualitatively.  Guinea-pigs  injected  with  such  mixtures 
sicken,  after  a  long  period  of  incubation,  with  typical  paralyses  and 
show  no  local  reaction.  The  hypothetical  toxic  constituent  which 
gives  rise  to  these  paralyse  sis  termed  "toxon." 

Why  then  is  it  impossible  to  demonstrate  the  action  of  the  toxon 
in  native  diphtheria  poison?  This  is  readily  explained  by  the  relative 
concentration  of  toxin  and  toxon  in  the  toxic  bouillon.  Quantitative 
analysis  has  shown  that  the  toxin  is  usually  much  more  (about  5  times) 
concentrated  than  the  toxon.  Hence  the  fractional  parts  of  the 
lethal  dose  which  allow  the  animal  to  live  long  enough  to  manifest 
toxon  effects  usually  contain  too  little  toxon  to  produce  the  typical 
paralyses.  If,  however,  a  large  amount  of  poison  is  so  far  neutralized 
with  serum  that  all  the  toxin,  with  the  higher  affinity,  is  just  bound 
and  the  toxon  is  still  free,  a  mixture  will  be  obtained  which  practically 
represents  a  pure  toxon  solution,  for  the  neutral  toxin-antitoxin 
molecules  play  no  role  in  an  animal  experiment.  It  is  at  once  appar- 
ent that,  in  view  of  the  individual  multiplicity  of  vital  phenomena, 
the  poisons  of  all  strains  of  diphtheria  bacilli  will  not  contain  both 
components  in  the  same  relative  concentration.  As  a  matter  of  fact, 
we  find  that  the  number  of  lethal  doses  contained  in  the  difference 
Lf  —  LQ  varies  enormously,  and  so  far  as  the  toxon  content  is  con- 
cerned the  variations  were  from  0  to  300%  figured  on  the  basis  of 
the  toxin  content.  It  will  be  well  to  enter  somewhat  more  into  a 
study  of  these  two  extremes,  for  these  striking  exceptions  to  the  typical 
conditions  argue  strongly  in  favor  of  the  views  here  presented.  One 
of  the  poisons  in  question  was  studied  by  Ehrlich,  and  was  remarkable 
in  that  the  difference  Lf  — L0  represented  only  1.7  lethal  doses.  We 
may  therefore  assume  that  the  poison  was  free  from  toxon  or  nearly 
so,  for  the  value  of  D  was  actually  quite  near  one  lethal  dose,  the 
figure  demanded  of  a  toxon-free  poison,  provided  toxin  and  anti- 
toxin combine  like  a  strong  base  with  a  strong  acid.  The  opposite 
extreme  was  manifested  by  a  poison  described  by  Dreyer  and  Madsen. 
The  constants  of  this  showed  that  it  contained  three  times  as  much 
toxon  as  toxin.  This  poison,  moreover,  gave  rise  to  toxon  effects 
when  sublethal  dose  of  the  native  poison,  without  serum  addition, 
were  injected  into  animals.  In  view  of  what  we  have  said  above, 
this  is  readily  understood,  the  relative  concentration  of  toxon  in 
this  case  was  so  great  that  even  sublethal  doses  sufficed  to  make  the 
toxon  effects  manifest.  In  most  native  poisons  this  demonstration 
fails  because  of  the  slight  relative  content  of  toxon. 


TOXIX   AND   ANTITOXIN:    METHODS  OF  THEIR  STUDY.    551 

The  existence  of  the  toxons  which  has  been  deduced  mathematic- 
ally from  the  biological  experiments  is,  however,  no  longer  based 
merely  on  these  calculations.  At  the  present  time  their  existence 
is  a  proven  fact,  for  quite  recently  van  Calcar  succeeded  in  separately 
isolating  toxin  and  toxon  from  the  native  poison  solution  by  means 
of  a  ingenious  dialyzing  procedure.  Owing  to  its  smaller  molecular 
volume,  toxin  diffuses  through  a  suitable  membrane  under  less  ten- 
sion than  toxon.  In  this  way  one  obtains  toxon-free  toxin  on  one 
side  and  toxin-free  toxon  on  the  other. 

This  direct  confirmation  of  the  conclusions  drawn  from  the  bio- 
logical analysis  of  the  toxins  shows  how  a  mathematical  study,  pro- 
vided biological  facts  are  carefully  regarded,  can  get  at  the  nature 
of  the  phenomena  in  question,  despite  the  failure  of  chemical  methods. 
To  be  sure  the  mathematical  treatment  of  biological  problems  must 
be  undertaken  very  carefully.  The  phenomena  of  animate  nature 
are  so  manifold,  and  subject  to  so  much  change,  that  they  cannot 
all  be  forced  into  the  limits  of  a  formula.  It  is  particularly  dangerous 
to  build  up  formulas  and  laws  on  the  basis  of  too  simple  assumptions. 
For  them  one  can  easily  be  deceived  by  the  apparent  exactness  of 
figures,  and  arrive  at  conclusions  which  do  not  sufficiently  regard  the 
complexity  of  the  actual  phenomena. 

Unfortunately  these  warnings  are  much  needed  at  the  present 
time,  for  certain  high  authorities  are  striving  energetically  to  explain 
the  most  complex  phenomena,  like  those  which  occur  in  the  union 
of  toxin  and  antitoxin,  as  though  they  were  simple  and  readily  cal- 
culated reactions  between  simple  substances. 

In  opposition  to  the  plurality  of  the  poison  constituents  demon- 
strated by  Ehrlich,  Arrhenius  and  Madsen,  as  is  well  known,  uphold 
a  Unitarian  standpoint.  Their  deductions  are  based  entirely  on 
the  method  of  partial  neutralization  introduced  into  toxin  study  by 
Ehrlich  and  referred  to  above.  Up  to  this  point  they  differ  only  in 
the  method  of  representing  their  results  graphically.  For  this  purpose 
they  use  a  system  of  coordinates,  laying  off  the  amounts  of  antitoxin 
contained  in  each  mixture  on  the  abscissas.  But  whereas  in  Ehrlich's 
scheme  the  ordinates  represent  the  amounts  of  toxin  which  each 
addition  of  antitoxin  causes  to  disappear,  Arrhenius  and  Madsen  use 
the  ordinates  to  represent  the  toxicity  which  each  mixture  still  retains. 
In  their  work  these  authors  observed  that  now  and  then  in  a  num- 
ber of  poisons,  especially  in  tetanolysin,  the  line  connecting  the  points 
plotted  possessed  a  certain  similarity  to  curves  obtained  when  weak 


552  COLLECTED   STUDIES  IN    IMMUNITY. 

bases  are  neutralized  by  weak  acids  (ammonia  and  boric  acid).  This 
similarity  constitutes  the  basis  for  their  mathematical  work,  which 
leads  them  to  conclude  that  toxin  and  antitoxin  are  simple  substances 
whose  reaction  is  reversible.  This  reaction  finds  its  expression  in 
the  curve  just  mentioned.  Let  us  examine  their  conclusions  and 
see  whether  they  are  justified. 

The  two  graphic  methods  referred  to  are  equally  correct.  Never- 
theless it  cannot  be  denied  that  the  one  employed  by  Ehrlich,  the 
so-called  " poison  spectrum,"  has  certain  advantages,  for  it  brings 
out  more  clearly  any  deviations  from  the  regular  curve.  Speaking 
mathematically  we  say  that  the  " poison  spectrum'7  is  the  graphic 
representation  of  the  differential  quotients  of  Arrhenius  and  Madsen's 
curve.  In  this  sense,  the  ordinates  of  the  spectrum  represent  the 
direction  of  the  neutralization  curve,  i.e.,  the  trigonometric  tangent 
of  the  angle  which  the  tangent  forms  at  every  point  with  the 
axis  of  the  abscissas.  Hence,  if  the  course  of  the  neutralization 
curve  is  that  of  a  straight  line,  the  direction  therefore  being  the  same 
at  all  points,  we  must  represent  the  poison  spectrum  as  a  rectangle. 
If,  as  is  often  the  case,  the  addition  of  a  small  amount  of  antitoxin 
causes  no  decrease  in  toxicity  (prototoxoids),  so  that  the  neutraliza- 
tion curve  in  this  part  of  its  course  lies  parallel  to  the  axis  of  the 
abscissas,  we  must  represent  the  poison  spectrum  as  having  a  gap 
at  this  point,  for  the  angle  between  tangent  and  axis  of  abscissas 
is  0°.  This  brief  statement  should  make  it  clear  that  in  the  poison 
spectrum,  by  representing  the  direction  of  the  separate  parts  of  the 
curve  as  ordinates,  deviations  from  the  regular  curve-like  course 
will  be  more  clearly  shown.  It  may  be  well  to  study  these  conditions 
by  means  of  a  diphtheria  poison  investigated  by  Madsen.1  See 
Figs.  1  and  2. 

These  figures  show  that  the  deviations  from  the  hyperbolic  curve 
demanded  by  Arrhenius  and  Madsen's  views  are  much  more  clearly 
shown  in  the  representation  employed  by  Ehrlich.  Entirely  aside 
from  the  question  whether  the  sharply  defined  zones  of  the  poison 
spectrum  actually  exist,  or  whether  a  gradual  transition  must  be  inter- 
polated, it  is  certain  that  the  changes  should  always  occur  in  the  same 
way;  for  they  merely  represent  the  differential  quotients  of  the 
neutralization  curve,  and  should  therefore,  if  this  curve  were  hyper- 
bolic, show  a  successive  decrease.  The  manifestly  very  irregular 

1  The  sole  object  in  employing  this  poison  is  to  illustrate  the  two  methods 
of  graphic  representation. 


TOXIX  AND  ANTITOXIN:    METHODS  OF  THEIR  STUDY.    553 

rise  and  fall  of  the  differential  quotients  shows  at  once  that  a  hyper- 
bolic curve  is  out  of  the  question  in  the  case  pictured  above.  If 
we  examine  the  poison  spectrum,  on  the  other  hand,  we  find  that  this 
represents  Madsen's  poison  entirely  in  accord  with  Ehrlich's  views 
concerning  the  constitution  of  diphtheria  poison.  If  toxin  and  anti- 
toxin unite  firmly,  and  the  course  of  the  neutralization  curve  there- 
fore is  a  straight  line,  the  irregular  course  is  explained  by  the  toxoid 
present  in  the  poison  and  by  the  varying  affinity  of  the  poison  con- 
stituents The  highest  zone  in  the  poison  spectrum  (zone  c)  indicates 
that  at  this  point  equal  amounts  of  antitoxin  cause  the  greatest. 


10 


a., 

PROTOTOJXOID 

&., 

HEMITOXIN 

c., 

PURE  TO 

IN 

TOXON 

)          0.05        0.1         0.15        0.2        0.25        0.3        0.35        0.4      ,  <fc 

FIG.  1. — Poison  spectrum  according  to  Ehrlich. 


decrease  in  toxicity.  Hence  this  part  of  the  poison  must  contain 
the  least  toxoids,  or  none  at  all,  and  we  may  therefore  speak  of  this 
as  pure  toxin.  It  will  serve  as  a  unit  for  judging  the  degree  of  con- 
tamination with  toxoid  in  the  remaining  portions.  We  should  then 
speak  of  zone  b  as  the  hemitoxin,  i.e.,  for  each  molecule  of  toxin 
there  is  one  of  toxoid.  The  sequence  of  the  different  zones  corre- 
sponds to  the  different  affinities  of  the  components.  Thus  we  see 
that  the  addition  of  a  small  amount  of  antitoxin  (a)  does  not  cause 
any  decrease  of  toxicity  whatever.  And  yet  the  antitoxin  must 
have  been  bound.  We  conclude,  therefore,  that  toxoids  must  here 
be  present  which  possess  a  higher  affinity  than  any  other  constituent 
of  the  poison.  We  are  here  dealing  with  the  important  prototoxoid 
zone  which  we  encounter  so  frequently  in  diphtheria  poison,  abrin, 
ricin,  crotin,  etc.  The  hemitoxin  zone  which  follows  this  is  to  be 
regarded  as  a  deuterotoxin  in  its  affinity.  The  constituents  of  tha 


554 


COLLECTED  STUDIES  IN   IMMUNITY. 


poison  can  thus  be  arranged  as  proto-,  deutero-, ,  tritotoxin,  etc., 

after  which  finally  comes  the  constituent  possessing  the  weakest 
affinity,  namely,  the  toxon.  That  this  varied  affinity  does  not  arise 
when  the  toxoids  are  formed,  but  differentiates  the  undecomposed 
constituents  of  the  poison  from  the  outset,  is  demonstrated  by 
the  genesis  of  toxoid  formation.  Thus  if  one  is  in  a  position  to 
study  a  very  pure  poison  in  its  various  stages  of  decomposition,  it  will 
be  found  that  there  is  a  first  phase  which  leads  to  the  formation  of 
hemitoxin,  and  that  a  later  phase  changes  this  into  pro^otoxoid. 
If  there  were  a  change  in  affinity, 
however,  we  should  have  had  a 
pure  toxoid  zone  from  the  start. 

The  prototoxoids  proved  a 
serious  obstacle  to  Arrhenius  and 
Madsen  in  the  logical  develop- 
ment of  their  views.  According  to 
their  theory  just  the  first  amounts  « 
of  antitoxin  added  should  c 
crease  the  toxicity  the  most,  jj 
Nevertheless  a  number  of  experi-  2 
ments  were  published  by  these 
authors  (Madsen,  with  diphtheria 
poison,  and  Madsen  and  Wal- 
baum,  for  ricin)  in  which  the  proto- 
toxoids and  their  development  were 
only  too  apparent.  And  Arrhenius 
and  Madsen  seem  to  appreciate 
that  they  can  no  longer  explain  this 
contradiction  by  assuming  that  the 
prototoxoid  zone  is  due  to  "  change-  FIG.  2.— Neutralization  curve  accord- 
ments  minimes  dans  le  milieu  am-  ing  to  Arrhenius  and  Madsen. 
biant,"  or  by  saying  that  the  proto- 
toxoid zone  is  "of  little  interest."  In  order,  therefore,  to  eliminate 
these  prototoxoids,  so  annoying  for  their  formula,  they  have  discarded 
the  well-tried  criterion  for  a  fatal  dose  of  diphtheria  poison  (death 
of  the  guinea-pig  in  3  to  4  days),  and  now  attempt  to  calculate  the 
fatal  dose  in  a  new  way.  Their  procedure  is  as  follows:  Retaining 
the  definition  of  a  fatal  dose,  they  believe  it  possible  to  calculate  the 
fraction  or  multiple  of  the  fatal  dose  employed,  from  the  time  of 
the  animal's  death  or  even  from  the  resulting  loss  of  weight.  Such 


60 
45 
40 
35 
30 
25 
20 
15 
10 
5 
0 

\ 

\ 

\ 

\ 

\ 

\ 

\ 

\ 

\ 

\ 

V, 

-N» 

TOXIN   AND  ANTITOXIN:    METHODS  OF  THEIR   STUDY.    555 

a  procedure,  in  order  to  possess  any  justification  whatever,  would 
have  to  be  based  on  an  enormous  experience.  But  even  aside  from 
this  it  is  amazing  to  see  how  a  lot  of  experimental  protocols,  going 
back  to  1897,  are  unhesitatingly  used  for  their  calculations.  The  old 
determinations  of  the  lethal  dose,  in  which  death  produced  acutely 
in  3  to  4  days  was  the  criterion,  are  very  difficult  to  make  use  of 
o.ving  to  the  individual  variations  in  the  animals.  Certainly  it  re- 
quires some  experience  to  know  which  animals  should  be  discarded 
because  of  over-  or  undersusceptibility.  But  how  much  more  com- 
plex the  conditions  really  are  is  at  once  apparent  if  one  attempts  to 
determine  £  or  ^  of  a  lethal  dose  from  the  clinical  course  of  the  disease. 
Hence  it  is  not  surprising  to  find  that  the  lethal  doses  calculated 
by  Arrhenius  and  Madsen  represent  the  averages  of  figures  which 
often  differ  from  each  other  by  many  times.  The  tedious  work 
which  these  authors  have  undertaken  may  perhaps  satisfy  a  mathe- 
matician, to  the  biologist,  however,  it  can  only  represent  useless 
and  dangerous  playing  with  figures.  It  signifies  nothing,  therefore, 
if  the  figures  recently  obtained  by  this  method  by  Arrhenius  and 
Madsen  with  three  poisons  fail  to  show  any  prototoxoid  zone.1  For 
the  same  reason,  also,  we  cannot  regard  certain  other  figures,  which 
differ  markedly  in  observation  and  calculation,  as  arguments  against 
their  views. 

However,  we  need  neither  confirmation  nor  controversion  of 
their  theory.  For  it  has  been  found  that  the  assumptions  on  which 
this  theory  is  based  have  no  existence  whatever.  We  have  already 
alluded  to  the  fact  that  van  Calcar  has  recently  demonstrated  the 
existence  of  toxons.  But  it  has  also  been  shown  by  another  method 
that  diphtheria  poison,  as  well  as  most  other  toxins,  must  contain 
various  constituents  capable  of  binding  the  antitoxin.  This  method 
had  its  inception  in  the  following  considerations. 

Arrhenius  and  Madsen,  as  already  stated,  regard  the  union  of  toxin 
and  antitoxin  as  a  reversible  reaction  between  two  simple  [einheitlich] 
substances.  According  to  this  view,  therefore,  the  reaction  is  incom. 
plete,  i.e.,  the  two  substances  reacting  (toxin  and  antitoxin)  are 
never  completely  used  up,  a  certain  portion  of  both  toxin  and  anti- 
toxin always  remaining  free  beside  the  neutral  toxin -antitoxin  combi- 
nation. The  equilibrium  which  exists  between  the  three  components 

1  We  should  not  neglect  to  mention  that  the  existence  of  the  prototoxoid 
^one  and  its  development  from  the  hemitoxin  phase  has  also  been  demonstrated 
in  diphtheria  poison  by  so  excellent  a  worker  as  Theobald  Smith. 


556  COLLECTED  STUDIES  IN   IMMUNITY. 

will  then  be  governed  by  the  law  of  mass  action  formulated  by 
Guldberg-Waage,  namely,  (toxin)  .(antitoxin)  =k (toxin-antitoxin),  in 
which  the  brackets  denote  the  concentration,  and  k  the  constant  of 
equilibrium  to  be  determined  for  each  poison.1  All  the  calculations 
of  Arrhenius  and  Madsen  are  based  on  this  formula,  and  their  entire 
work  stands  or  falls  with  the  applicability  of  the  formula  to  the  sub- 
ject of  toxins. 

The  formula,  however,  is  only  then  applicable  if  the  reaction 
is  really  completely  reversible,  and  this  is  not  the  case.  Thus  if  mix- 
tures containing  the  same  amounts  of  toxin  and  antitoxin  are  tested 
at  the  end  of  the  reaction,  it  is  easy  to  convince  one's  self  that  the 
toxicity  is  dependent  not  only  on  the  amounts  of  toxin  and  anti- 
toxin, but  on  the  manner  of  making  the  mixtures.  If  to  the  same 
amount  of  antitoxin  we  add  at  intervals  fractional  parts  of  the  toxin, 
we  shall  find  that  the  resulting  end  product  is  considerably  more  toxic 
than  if  the  same  amount  of  toxin  is  mixed  with  the  antitoxin  at  once. 
This  holds  true  even  if  the  toxin  is  added  at  the  time  corresponding 
to  the  addition  of  the  last  fraction  in  the  former  case.  Von  Dungern 
was  the  first  to  point  out  the  significance  of  this  experiment,  in  con- 
nection with  an  observation  made  by  Danysz,  for  the  question  of 
reversibility.  He  showed  that  if  this  really  was  a  completely  reversible 
reaction  between  simple  substances,  as  is  assumed  by  Arrhenius  and 
Madsen,  we  should  expect  that  the  same  equilibrium  should  always 
ensue  with  the  same  total  amounts  of  reacting  substances,  i.e.,  the 
toxicity  of  the  end  products  should  always  be  the  same.  Any  devia- 
tion from  this  could  occur  in  the  fractioning  process  only  during  the 
course  of  the  reaction ;  and  then,  provided  the  deviation  were  a  function 
of  the  reaction-time,  this  would  be  just  the  reverse  of  what  is  actually 
observed.2  Hence  all  those  poisons  in  which  this  phenomenon  of 

1  In  their  recent  publications  Arrhenius  and  Madsen  assume  that  one  mole- 
cule toxin  combines  with  one  molecule  antitoxin,  not  to  form  two  molecules 
of  the  toxin-antitoxin  combination,  as  the  above  formula  would  show,  but  that 
two  different  substances  are  formed,  toxinan  and  titoxin.     To  be  sure  as  the 
equation  then  reads,  (toxin)   (antitoxin )=  k  (toxinan)   (titoxin),  one  objection 
to  the  above  formula  is  done  away  with,  but  a  new  hypothesis,  lacking  all  evi- 
dence whatever,  is  thus  introduced  merely  for  the  sake  of  the  formula. 

2  The  phenomenon  in  question  therefore  shows  exactly  the  reverse  of  what 
Arrhenius  and  Madsen's  theory  demand.     For  this  reason  the  limit  of  error 
need  not  be  considered,  although,  owing  to  the  enormous  quantitative  differences, 
it  would  play  no  role  in  judging  the  result.     Nor  can  Arrhenius  extricate  him- 
self from  the  predicament  by  suggesting  that  we  are  dealing  with  slowly  progress- 


TOXIN   AND  ANTITOXIN:    METHODS  OF  THEIR  STUDY.    557 

increasing  toxicity  on  the  fractional  addition  of  toxin  can  be  demon, 
strated  must  at  once  be  excluded  from  any  mathematical  analysis 
based  on  a  formula  of  equilibrium  derived  from  the  law  of  mass  action. 
In  all  of  the  cases  l  examined  for  the  purpose  (diphtheria  poison, 
tetanolysin,  ricin,  staphylolysin,  arachnolysin,  rennin,and  precipitin), 
this  method  has  shown  that  the  conception  of  Arrhenius  and  Madsen 
is  entirely  inapplicable. 

The  phenomena  observed,  however,  are  very  readily  explained 
by  the  assumption  of  a  plurality  of  combining  groups  in  the  poison 
solution.  Thus  if  to  an  excess  of  antitoxin  a  small  quantity  of 
poison  is  added,  as  is  done  in  the  fractioning  experiment,  the  result 
would  be  that  even  the  constituents  possessing  a  feeble  affinity  and 
which  are  of  no  consequence  so  far  as  any  toxic  action  is  concerned, 
would  be  bound  by  the  antitoxin.  When  then  the  second  portion 
of  poison  is  added,  it  will  be  impossible  for  the  toxin  molecules,  al- 
though possessing  a  higher  affinity,  to  crowd  the  previously  bound 
constituents  out  of  their  combination  with  the  antitoxin.  The  result 
is  that  a  certain  portion  of  toxin,  which  would  have  been  neutralized 
by  the  antitoxin  if  all  the  poison  had  been  mixed  with  the  antitoxin 
at  once,  now  remains  free.  That  is  to  say,  the  fractional  method  of 
adding  the  poison  has  resulted  in  an  increased  toxicity,  the  Lf  dose 
being  reached  with  a  smaller  amount  of  poison.  Furthermore  it  is 
possible,  by  means  of  suitable  technique,  to  cause  a  reduction  of  the 
L0  dose,  from  which  it  follows  that  the  L0  serum  mixture  contains 
free  non-toxic  constituents  capable  of  binding  antitoxin,  and  that 
these  must  possess  still  less  affinity  than  the  toxon.  These  are  the 
so-called  "  epitoxonoids "  of  von  Dungern.  The  discovery  of  the 
epitoxonoids  also  offers  an  easy  explanation  of  the  fact  that  it  is 
possible  to  immunize  with  mixtures  of  toxin  and  antitoxin  which  are 
physiologically  neutral. 

All  this  shows  that  a  complete  reversibility,  even  of  the  individual 


ing  side  reactions  which  do  not  interfere  with  the  main  reaction  when  one  works 
rapidly.  For,  as  was  pointed  out  by  von  Dungern  and  Sachs,  the  increased 
toxicity  is  already  demonstrable  at  a  time  when  the  union  of  toxin  and  antitoxin 
is  not  yet  ended.  The  hypothetical  "side  reaction"  would  therefore  proceed 
just  as  quickly  as  the  main  neutralizing  reaction. 

1  The  single  exception  met  with,  namely  cobra  venom,  only  proves  the  rule; 
for  cobra  venom  (we  are  dealing  with  the  hsemolytic  portion  which  is  activated 
by  lecithin)  is  a  simple  substance  with  a  strong  affinity  for  the  antitoxin,  as  can 
be  seen  from  the  course  of  the  neutralization  curve,  which  is  a  straight  line. 


558  COLLECTED  STUDIES   IN  IMMUNITY. 

poison  constituents,  is  out  of  the  question.  On  the  contrary  we  must 
assume  that  the  union  of  these  substances  with  the  antitoxin  is  subse- 
quently tightened.  This  tightening  is  also  borne  out  by  other  observa- 
tions, both  old  and  recent.  If  the  toxin-antitoxin  reaction  were 
reversible,  it  should  be  possible,  by  removing  the  supposedly  free 
toxin  residue,  to  constantly  change  the  equilibrium,  so  that  the  toxin 
could  all  be  recovered.  Nevertheless,  although  toxin  can  be  filtered 
through  gelatine  and  antitoxin  cannot,  it  is  impossible  either  by 
gelatine  filtration  (Martin  and  Cherry)  or  by  gelatine  diffusion  (van 
Calcar)  to  obtain  free  toxin  from  neutral  toxin-antitoxin  mixtures.1 
In  addition  to  this  one  cannot  help  being  surprised  that  the  calcula- 
tions of  Arrhenius  and  Madsen  entirely  ignore  the  cells'  toxin-binding 
receptors  which  effect  the  poisoning.  In  accordance  with  their 
views,  these  receptors  should  represent  an  important  element  in  the 
equilibrium;  and  yet  they  appear  to  have  entirely  overlooked  this 
fact. 

It  would  lead  us  too  far  to  discuss  all  the  arguments  against  the 
views  of  Arrhenius  and  Madsen.  It  will  suffice  to  call  attention  to 
the  serious  objections  which  Nernst  has  raised  regarding  the  prin- 
ciples involved,  and  to  Koppe's  criticism  of  their  technique  in  making 
hsemolytic  test-tube  experiments.  This  illustrates  the  danger  of  a 
one-sided  mathematical  study  of  biological  problems.  Even  if  one 
succeeds  now  and  then  in  making  the  figures  of  observations  and  cal- 
culation tally,  it  is  impossible  at  the  present  time  for  these  mathe- 
matical expressions  to  explain  the  facts.  To  be  sure  they  may  be 
able  to  represent  the  resultants  of  the  processes  which  bring  about 
the  phenomena,  but  in  that  case  the  formula  is  nothing  more  than 
an  interpolation  formula.  Corresponding  to  this,  therefore,  we  see 
that  the  formulas  of  Arrhenius  and  Madsen  vary  widely  for  the  same 
poison,  every  new  lot  of  poison  of  the  same  bacillary  origin  has  a  new 
constant  of  equilibrium.  Hence  the  formula  is  applicable  only  to 
one  particular  case,  and  so,  even  if  it  were  a  correct  interpolation 
formula,  progress  of  biological  science  would  in  no  way  be  furthered 
by  it. 

1  It  is  perfectly  evident  that  toxin  can  be  obtained  from  fresh  toxin-antitoxin 
mixtures  by  diffusion  through  gelatine,  and  this  has  recently  been  demonstrated 
by  Madsen  and  Walbaum.  According  to  Morgenroth  such  mixtures  require 
at  least  twenty-four  hours  for  the  union  to  become  complete.  Hence  the  state- 
ment by  Madsen  and  Walbaum  that  the  mixtures  must  be  fresh  in  order  to 
demonstrate  what  they  regard  as  dissociation  only  confirms  our  view. 


TOXIX   AND  ANTITOXIN:    METHODS   OF  THEIR  STUDY     559 

Biology  does  not  content  itself  with  a  mere  registration  of  phe- 
nomena; it  seeks  to  discover  their  nature  and  their  relation  to  one 
another.  In  fact  the  chief  mission  of  biology  is  to  attempt,  by  link- 
ing facts  and  theories  and  hypotheses,  to  satisfy  the  craving  of  the 
thinking  naturalist  for  an  insight  into  causes. 


The  following  is  a  summary  of  the  literature  bearing  on  this  subject. 

1897.  P.  EHRLICH,  Die  Werthbemessung  des  Diptherieheilserums.     Klinisches 

Jahrbuch. 

The    same,    Zur    Kenntniss    der    Antitoxinwirkung.     Fortschritte    der 
Medizin. 

1898.  The  same,  Cber  die  Constitution  des  Diphtheriegiftes.     Deutsche  med. 

Wochenschrift. 

1899.  TH.  MADSEN,  Uber  Tetanolysin.     Zeitschr.  fur  Hygiene,  Vol.  32. 

1902.  S.  ARRHEXIUS  AND  TH.  MADSEN,  Physical  chemistry  applied  to  toxins 

and  antitoxins.     Festsknit  ved  Indvielsen  af  Statens  Serum  Institut. 

1903.  The  same,  Anwendung  der  physikalischen  Chemie  auf  das  Studium  der 

Toxine  und  Antitoxine.     Ztschr.  f.  physik.  Chemie,  Vol.  44. 
P.   EHRLICH,    Uber  die   Giftcomponenten   des   Diphtherietoxins.     Berl. 

klin.  Wochenschr. 
E.    VON    DUNGERX,    Bindungsverhaltnisse    bei    der    Pracipitinreaktion. 

Centralblatt  f.  Bacteriol.,  Vol.  34. 
TH.   MADSEN.    La   constitution    du   poison   diphthe"rique.     Centralblatt 

f.  Bacteriol.,  Vol.  34. 

1904.  S.    ARRHENIUS,    Die   Anwendung   der    physikalischen   Chemie   auf   die 

Serumtherapie.     Arbeiten  a.  d.  Kaiserl.  Gesundheitsamte,  Vol.  20. 
The    same,   Zur    Theorie    der    Bindung    von    Toxin     und    Antitoxin. 

Berlin,  klin.  Wochenschr. 
P.  EHRLICH,  Bemerkungen  zur  Mitteilung  von  Arrhenius:    Zur  Theorie 

der  Absattigung  von  Toxin  und  Antitoxin.     Berl.  klin.  Wochenschr. 
E.  VON  DUNGERN,  Beitrag  zur  Kenntniss  der  Bindungsverhaltnisse  bei 

der  Vereinigung  von  Diphtheriegift  und  Antiserum.     Deutsch.  med. 

Wochenschr. 
S.    ARRHEXIUS,    Die    Anwendung   der   physikalischen    Chemie   auf   die 

serumtherapeutischen  Fragen.     Boltzmann  Festschrift. 
H    SACHS,   Uber   die  Constitution   des   Tetanolysins.       Berl.  klin.  Wo- 
chenschr. 

P.  KYES,  Cobragift  und  Antitoxin.     Berliner  klin.  Wochenschrift. 
TH.  MADSEN  et  L.  WALBAUM.  Toxines  et  Antitoxines.     De  la  ricine  et 

de  1'antiricine      Centralblatt  f.  Bacteriol.,  Vol.  36. 
W.  NERXST,  Uber  die  Anwendbarkeit  derGesetze  des  chemischen  Gleich- 

gewichts  auf  Gemische  von  Toxin  und  Antitoxin.     Ztschr.  f.  Electro- 

chemie,  Vol.  X,  No.  22. 


560  COLLECTED  STUDIES  IN   IMMUNITY, 

J.   MOKGENROTH,   Untersuchungen   iiber  die    Bindung  von    Diphtherie- 

toxin  und  Antitoxin,  sowie   iiber  die   Constitution  des   Diphtherie- 

giftes.     Berlin,  klin.  Wochenschr.    In  detail  in  Zeitschr.  f.  Hygiene, 

Vol.  48. 
H.  KOPPE,  Zur    Anwendung   der   physikalischen    Chemie   auf   das 

Studium  der  Toxine  and  Antitoxine  und  das  Lackfarbenwerden 

rother  Blutscheiben.     Pfliiger's  Archiv,  Vol.  103. 
An  address  entitled  "Die  Serumtherapie  vom  physikalisch-chemischen 

Standpunkte,"  by  Sv.  Arrhenius.     Discussion  by  Ehrlich,  Nernst, 

Arrhenius.     Zeitschr.  f.  Electrochemie,  Vol.  X,  No  35. 
E.  VON  DUNGERN,  Bemerkung  zum  Vortrag  von  Professor  S.  Arrhenius: 

"Die  Serumtherapie  vom  physikalisch-chemischen  Standpunkte." 

Zeitschr.  f.  Electrochemie,  Vol.  X,  No.  40. 

TH.  MADSEN,  Toxins  and  Antitoxins.     British  Medical  Journal. 
R.  P.  VAN  CALCAR,  Uber  die  Constitution  des  Diphtheriegiftes.     Berl. 

klin.  Wochenschr. 
S.  ARRHENIUS  et  TH.   MADSEN,   Toxines  et  Antitoxines.     Le  Poison 

diphtherique.     Centralblatt  f.  Bacteriol.,  Vols.  36  and  37. 
H.  SACHS,  l)ber  die  Bedeutung  des  Danysz-Dungernschen  Kriterium 

nebst  Bemerkungen  uber  Prototoxoide.     Centralblatt  f.  Bacteriol. » 

Vol.  37. 
L.  MICHAELIS,  A  collection  of  studies  on  this  question  together  with  a 

critical  review.     Biochemisches  Centralblatt,  Vol.  VI,  No.  1. 


XL.    THE    MECHANISM   OF  THE   ACTION   OF 
ANTI AMBOCEPTORS .' 

By  Prof.  PAUL  EHRLICH  and  Dr.  H.  SACHS. 

OWING  to  closer  investigations  into  the  nature  of  immunity  our 
conceptions  regarding  the  relation  between  antibody  and  the  sub- 
stances exciting  the  production  of  immunity  (the  antigen,  as  it  is 
called)  have  undergone  a  certain  modification.  This  consists  in  a 
more  precise  definition  of  the  concept  specificity.  In  the  beginning 
it  was  assumed  that  an  antibody  produced  by  immunization  acted 
only  against  the  substance  through  which  it  was  developed.  Further 
observations,  however,  soon  brought  to  light  cases  in  which  this 
law  was  apparently  violated.  A  clear  insight  into  this  subject  was 
finally  made  possible  when  the  receptor  was  looked  upon  as  the 
agent  which  excited  the  production  of  immunity.  According  to 
the  side-chain  theory,  therefore,  specificity  of  the  antibodies  always 
means  "  the  specific  relations  between  the  individual  types  of  antibodies 
and  of  receptors."  2  Since,  therefore,  the  same  receptor  can  be  dis- 
tributed not  only  among  different  kinds  of  cells,  and  bodies  of  differ- 
ent functions  all  within  the  same  animal  species,  but  also  among 
different  species  of  animals,  we  see  that  it  is  impossible  to  speak  of 
a  specificity  in  a  zoological  sense,  or  of  a  specificity  in  respect  to  the 
morphological  or  functional  properties  of  the  antigens.  The  anti- 
body is  specific  only  for  the  receptor,  i.e.,  for  those  elements  possess- 
ing this  fitting  receptor. 

Of  the  various  substances  W7hich  excite  the  production  of  im- 
munity, a  special  place  is  occupied  by  the  receptors  of  the  third  order: 
these,  when  free,  constitute  the  amboceptors.  As  is  well  known, 
the  amboceptors  possess  a  double  function.  On  the  one  hand  they 
unite  with  the  cytophile  group  of  the  cells,  and  on  the  other  with  the 

1  Reprinted  from  Berliner  klin.  Wochenschrift,  1905,  No.  19. 
a  P.  Ehrlich  and  Morgenroth,  Hsemolysins.     See  page  88. 

561 


562  COLLECTED  STUDIES  IN  IMMUNITY. 

complementophile  group  of  the  complement.  Each  of  these  two 
haptophore  groups  will  therefore  be  able  to  excite  the  production 
of  corresponding  antibodies,  a  fact  to  which  attention  was  called 
in  the  Croonian  lecture,  1900.1 

"The  lysin,  be  it  bacteriolysin  or  hsemolysin,  possesses  altogether 
three  haptophore  groups,  of  which  two  belong  to  the  immune  body 
and  one  to  the  complement.  Each  of  these  haptophore  groups  can 
be  bound  by  an  appropriate  antigroup."  Three  '  antigroups '  are  thus 
conceivable,  any  one  of  which,  by  uniting  with  one  of  the  haptophore 
groups  of  the  lysin,  can  frustrate  the  action  of  the  lysin." 

In  other  words,  according  to  the  amboceptor  theory  two  different 
antiamboceptors  are  at  once  conceivable,  either  of  which  would 
inhibit  the  action  of  the  amboceptor.  One  would  act  by  preventing 
the  union  of  amboceptor  and  cell,  the  other  by  preventing  the  comple- 
ment from  uniting  with  the  amboceptor.  Originally  the  antiambo- 
ceptors produced  by  immunization  were  regarded  as  being  directed 
against  the  cytophile  group.2  In  view  of  this  it  was  extremely  de- 
sirable for  the  support  of  the  amboceptor  theory  that  the  existence 
of  antibodies  for  the  complementophile  group  should  be  demon- 
strated. This  has  recently  been  done  by  Bordet,3  and  it  is  strange 
to  see  that  he  employs  his  discovery  in  combating  the  receptor  theory 
when  it  really  is  a  very  neat  confirmation  of  this. 

Bordet  finds  that  antiamboceptors  can  be  produced  not  only  by 
immunization  with  hsemolytic  immune  serum,  but  also  with  normal 
serum  of  the  same  species,  even  though  this  normal  serum  contains 
no  corresponding  amboceptors.  He  treated  guinea-pigs  with  normal 
rabbit  serum  which  contains  no  hsemolytic  amboceptors  for  ox 
blood,  and  obtained  an  immune  serum,  which  yet  was  able  to  in- 
hibit the  action  of  the  amboceptors  derived  by  immunizing  with 
ox  blood.  That,  certainly,  is  a  discovery  which  cannot  readily  be  ex- 
plained in  harmony  with  Bordet's  sensitization  theory.  According 
to  Bordet,  as  we  know,  these  immune  bodies  (his  "sensitizers") 
possess  the  one  property  of  combining  with  the  susceptible  cell 
and  thus  rendering  this  vulnerable  to  the  action  of  the  complement. 
This  being  the  case  it  is  incomprehensible  how  a  serum  which.possesses 

1  P.  Ehrlich,  On  Immunity,  Proceedings  Royal  Society,  1900. 

2  Ehrlich  and  Morgenroth,  VI.  Communication,  page  88. 

3  J.  Bordet,  Les  propriete's  des  antisensibilatrices   et  les  theories  chimiques 
de  l'immunite\     Annal.  de  1'Instit.  Pasteur,  1904,  No.  10. 


MECHANISM  OF  THE  ACTION  OF  ANTIAMBOCEPTORS.      563 

no  sensitizers  whatever  for  the  species  of  cell  in  question  can 
yet  excite  the  production  of  antibodies  directed  against  them. 
The  matter  takes  on  an  entirely  different  aspect  if  we  regard  this 
phenomenon  from  the  standpoint  of  the  amboceptor  theory.  Ac- 
cording to  what  has  been  said  above  we  at  once  see  that  two  func- 
tionally different  types  of  antiamboceptors  are  possible.  In  Bordet's 
case  the  normal  rabbit  serum  possessed  no  amboceptors  (i.e.,  no  cyto- 
phile groups)  for  ox  blood;  therefore  the  antibodies  which  are  de- 
veloped cannot  be  antiamboceptors  directed  against  the  cytophile 
groups.  Hence  by  exclusion  one  will  already  pronounce  them  anti- 
ambocepturs  of  the  complementophile  group.  The  facts  brought  for- 
ward by  Bordet  all  go  to  confirm  this. 

If  such  antiamboceptors  are  to  be  produced,  the  only  requisite 
is  that  the  serum  used  for  immunization  must  contain  the  corre- 
sponding complementophile  groups.  Is  this  the  case  in  normal 
rabbit  serum?  Every  normal  rabbit  serum,  as  Bordet  admits,  con- 
tains a  large  number  of  different  amboceptors.  If,  by  immunizing 
with  a  given  species  of  cell,  a  new  specific  amboceptor  develops  in 
the  serum,  the  new  element  in  the  receptor  apparatus  is  really  only  the 
cytophile  group,  which  is  produced  in  response  to  immunization.  The 
complementophile  apparatus  need  not  suffer  the  least  change  quali- 
tatively; in  fact  according  to  our  conception  it  usually  does  not 
change  markedly,  there  is  merely  an  increase  in  the  complemento- 
phile groups  corresponding  to  the  formation  of  the  additional  immune 
body.  We  have  already  expressed  this  opinion  in  a  previous  paper.1 
"In  my  judgment  we  shall  arrive  at  a  correct  conception  if  we  pro- 
ceed from  the  standpoint  that  in  general  the  specific  amboceptors 
exhibit  a  uniform  structure  so  far  as  their  complementophile  portion 
is  concerned,  while  their  cytophile  groups,  which  physiologically 
are  concerned  with  the  absorption  of  food,  differ  most  widely." 

It  must  not  be  thought  that  this  uniform  constitution  of  the  com- 
plementophile portion  2  contradicts  the  assumption  of  a  multiplicity 

1  P.  Ehrlich,  Betrachtungen  iiber  den  Mechanismus  der  Amboceptorwirkung 
und  seine  teleologische  Bedeutung.     Koch  Festschrift,  Jena,   1903. 

2  For  the  present  we  cannot  say  whether  the  complementophile  complex 
is  really  uniform  throughout  or  whether,  perhaps,  certain  partial  groups  do 
not  differ  in  the  individual  amboceptor  types  of  the  same  animal  species.     Such 
a  condition  is  easily  conceivable.     In  any  event  we  must  assume  that  the  com- 
plementophile apparatus  of  the  amboceptors  of  a  given  species  is  identical 
at  least  in  some  essential  part  of  its  haptophore  functions,  and  that  this  char- 
acterizes it  as  coming  from  the  animal  species  in  question. 


564  COLLECTED  STUDIES  IN  IMMUNITY. 

of  complements.  Naturally  the  different  complements  must  have 
different  complementophile  groups  corresponding  to  them.  But,  as 
was  stated  in  the  Sixth  Communication  on  HaBmolysins,1  an  immune 
body,  in  addition  to  a  particular  cytophile  group,  contains  two,  three, 
or  more  complementophile  groups.  In  a  later  paper  Ehrlich  and 
Marshall  offered  experimental  evidence  for  just  this  point;  besides 
this,  Bordet's  experiments,  according  to  which  an  amboceptor  after 
having  combined  with  cellular  elements  is  able  almost  completely 
to  rob  a  serum  of  its  complement,  also  support  this  view.2 

We  must  therefore  conceive  the  amboceptor  to  be  structurally 
a  polyceptor,  and  assume  further  that  the  amboceptors  of  a  distinct 
species  are  all  supplied  with  a  large  number  of  complementophile 
groups  which  vary  considerably  in  detail  but  in  their  entirety  repre- 
sent a  uniform  complex.  This  complex  is  reproduced  in  all  the 
amboceptors  of  the  same  serum.  In  general  the  amboceptors  are 
different  and  specific  only  so  far  as  the  cytophile  group  is  concerned. 

This  being  so  it  will  at  once  be  clear  that  antiamboceptors  directed 
against  the  complementophile  groups,  and  obtained  through  immuni- 
zation with  any  particular  amboceptor,  will  act  against  all  ambocep- 
tors of  the  same  animal  species  no  matter  whether  these  ambo- 
ceptors are  normally  present  in  the  serum  or  have  been  produced 
by  immunization.  For  the  complementophile  amboceptor  apparatus 
is  the  same  for  all  types  of  amboceptors  of  the  same  species.  As  a 
result  of  this,  an  immune  serum  obtained  through  immunization 
with  normal  serum  contains,  thanks  to  the  normal  amboceptors  in 
the  serum,  antiamboceptors  directed  against  the  artificially  produced 
amboceptors  of  the  same  species.  This  explains  also  the  earlier 
observations  made  by  Pfeiffer  and  Friedberger  3  that  antiamboceptors 
obtained  by  immunizing  with  cholera  serum  act  also  against  typhoid 
serum;4  it  also  explains  the  recent  experiments  made  by  Bordet.  We 

1  Ehrlich  and  Morgenroth.     See  page  88. 

2  P.  Ehrlich  and    H.   T.  Marshall,  Uber  die  complementophilen    Gruppen 
der  Amboceptoren.     Berl.  klin.  Wochenschr.  1902,  No.  25. 

3  R.  Pfeiffer  and  E.  Friedberger,  Weitere  Beitrage  zur  Frage  dor  Antisera 
und  deren  Beziehungen  zu  den  bacteriolytischen  Amboceptoren.     Centralblatt 
f.  Bacteriol.  1904,  Vol.  37;    also  1903,  Vol.  34. 

4  Naturally  the   statement   made   by  Ehrlich  and   Morgenroth    (Berl.  klin. 
Wochenschr.  1901,  No.  21)  that  "it  seems   improbable,  unless   in  a  given  case 
a  fortunate  coincidence  intervenes,  that  anti-immune  bodies  will  be  obtained 
directed  against  the  bactericidal  immune  bodies"  cannot  apply  to  the  antiambo- 
ceptors directed  against  the  complementophile  groups.     That  statement  applies 


MECHANISM  OF  THE  ACTION  OF  ANTIAMBOCEPTORS.     565 

must  call  particular  attention  to  the  fact  that  the  chief  point  in 
Bordet's  study,  the  non-specificity  of  the  antiamboceptors  so  far  as 
the  cytophile  group  is  concerned,  had  already  been  published  by 
Pfeiffer  and  Friedberger.  These  authors  have  explained  the  fact 
entirely  in  accordance  with  our  views,  as  follows: 

"We  are  inclined  to  believe  that  the  various  immune  bodies  of 
one  and  the  same  animal  species  possess  one  group  in  common  which 
in  a  way  stamps  them  as  coming  from  that  particular  animal  organism. 
The  antiserum  must  possess  certain  relations  to  this  group."  To 
this  we  would  add  that  for  the  present  it  seems  simplest  to  class  this 
group  or  groups,  specific  for  the  animal  species,  with  the  complemento- 
phile  group.  In  the  amboceptor  we  differentiate  a  specific  cytophile 
group  and  a  large  apparatus  made  up  of  complementophile  groups. 
Aside  from  the  property  of  anchoring  the  cells,  the  latter  groups 
exercise  all  the  remaining  functions  of  the  amboceptor.  Considering 
that  the  normal  amboceptors  and  those  produced  by  immunization 
are  essentially  similar  (a  point  which  we  have  always  emphasized), 
it  is  perfectly  obvious  that  one  can  produce  the  same  antiamboceptors 
by  immunizing  with  normal  amboceptors.  Hence  what  Bordet's 
study  really  brings  forward  is  the  actual  experimental  demonstration 
of  what  we  had  long  expected  was  the  case. 

Naturally  we  were  able  to  confirm  all  of  Bordet's  statements  of 
fact.  We  had  at  our  disposal  the  serum  of  a  goat  which  had  been 
immunized  with  normal  rabbit  serum,  and  could  easily  convince 
ourselves  that  this  serum  acts  as  an  antiamboceptor  against  ambo_ 
ceptors  derived  from  rabbits  by  specifically  immunizing  with  ox 
blood.  Furthermore,  we  succeeded,  by  adding  the  antiamboceptor 
to  previously  sensitized  blood-cells,  to  protect  these  against  haemolysis 
by  complement.  The  antiamboceptor  acts  just  like  a  complementoid 
according  to  the  conception  of  "complementoid-blocking"  described 
by  one  of  us  some  time  ago.1  It  occupies  the  complementophile 
groups  and  so  prevents  the  anchoring  of  the  complement.2 

only  to  the  antibodies  directed  against  the  cytophile  groups,  since  it  is  to  be 
assumed  that  these  cytophile  groups,  which  have  their  natural  counter-groups 
in  bacterial  cells,  will  not  have  these  in  the  cells  of  higher  animals.  This  limi- 
tation, however,  does  not  apply  to  the  antiamboceptors  acting  on  the  comple- 
mentophile complex.  This,  then,  disposes  of  Bordet's  objections  to  this  point. 

1  Ehrlich  and   Sachs,   "Uber  den  Mechanismus  der  Amboceptorenwirkung. 
Berl.  klin.  Wochenschrift,  No.  21,  1902. 

2  We  must  not  fail  to  mention  that,  in  contrast  to  Bordet,  we  made  these  experi- 
ments without  the  addition  of  inactive  guinea-pig  serum,  and  were  able,  despite 


566  COLLECTED  STUDIES  IN  IMMUNITY. 

We  were  also  able  to  readily  confirm  Bordet's  statement  that  the 
antiamboceptor  action  is  easily  inhibited  by  normal  rabbit  serum, 
Naturally  the  normal  amboceptors,  whose  complementophile  groups 
excited  the  production  of  the  antiamboceptor,  will  combine  with 
this  antiamboceptor  and  so  be  able  to  deflect  it  from  the  amboceptor 
acting  in  the  given  case.  Since  we  regard  the  antiamboceptor  in 
the  sense  of  a  complementoid,  this  phenomenon  corresponds  in  prin^ 
ciple  to  that*  described  by  Neisser  and  Wechsberg  as  deflection  of 
complement.1 

The  entire  complex  of  phenomena  just  discussed  shows  most 
strikingly  that  our  assumption  harmonizes  best  with  the  observed 
facts.  We  assume  that  in  Bordet's  antiamboceptors  we  are  dealing 
with  antibodies  directed  against  the  complementophile  groups.  The 
existence  of  such  antiamboceptors  again  demonstrates  that  the 
amboceptor  theory  is  correct.  According  to  Bordet's  sensitization 
theory  only  such  antiamboceptors  are  conceivable  which  prevent 
the  amboceptor's  union  with  the  cell.  But  if  there  are  other  kinds 
of  antiamboceptors,  as  the  findings  just  discussed  show,  we  must 
assume  that  the  amboceptor  has  other  affinities  besides  those  for  the 
cell,  and  this  leads  us  at  once  to  the  conception  which  we  have 
defined  under  the  name  amboceptor.  The  sensitization  theory  must 
therefore  be  abandoned. 

The  next  question  which  arises  is  whether  or  not  it  is  possible 
by  means  of  immunization  with  amboceptors  to  produce  antiambo- 


this,  to  effect  an  inhibition  of  haemolysis  by  subsequently  adding  antiambo- 
ceptor. It  seems  to  us  that  this  simplified  procedure  is  more  convincing,  for 
it  will  hardly  be  claimed  that  the  guinea-pig  serum  is  a  better  suspending  medium 
than  physiological  salt  solution,  and  that  it  therefore,  in  contrast  to  the  latter, 
leaves  the  blood-cells  intact.  Furthermore,  inactive  guinea-pig  serum  itself 
inhibits  the  hasmolysis  of  ox  blood  by  amboceptor  and  complement  (guinea- 
pig).  Hence  when  guinea-pig  serum  is  present  the  question  whether  the  ab- 
sence of  haemolysis  is  due  to  an  antiamboceptor  or  not  is  left  undecided. 

1  In  contrast  to  Bordet,  however,  we  were  unable  by  means  of  normal  ambo- 
ceptor to  effect  the  subsequent  breaking  of  the  union  between  antiamboceptor 
and  sensitized  blood-cells.  It  may  be  that  in  our  case  the  union  between  anti- 
amboceptor and  sensitized  cells  so  rapidly  became  firm  that  it  could  no  longer 
be  dissolved  by  the  normal  amboceptor.  Even  Bordet  admits  that  this  dis- 
solution can  be  effected  only  for  a  certain  period,  and  that  then  the  union 
becomes  very  firm.  We  are  pleased  to  note  that  Bordet  accepts  this  conception 
of  a  gradual  tightening  of  the  union  of  these  substances,  a  conception  of  the 
highest  importance  in  the  study  of  immunity  reactions. 


MECHANISM  OF  THE   ACTION   OF  ANTIAMBOCEPTORS.     567 

ceptors  also  against  the  cytophile  group.  We  have  therefore  ex- 
amined another  antiamboceptor  serum,  and  compared  its  properties 
with  those  of  the  antiserum  made  by  injections  of  normal  rabbit 
serum.  This  serum,  like  the  latter,  was  also  obtained  from  a  goat, 
but  instead  of  using  normal  rabbit  serum  for  immunization  the  goat 
had  been  treated  with  the  serum  of  a  rabbit  previously  immunized 
with  ox  blood.  Our  experiments,  however,  did  not  permit  of  a 
decision  on  this  point.  We  are  unable  to  say  whether  among  the 
antiamboceptors  excited  by  the  injections  of  the  immune  serum  there 
were  any  directed  against  the  cytophile  group.  It  is  entirely  con- 
ceivable that,  despite  the  presence  of  the  cytophile  group,  these  are 
unable  to  exert  any  immunizing  power,  since  the  complementophile 
groups  invariably  encounter  the  corresponding  coufcter-group  in  the 
organism  and  so  are  the  only  ones  bound  to  the  tissue  receptors.  In 
that  case  previous  to  injection  one  would  attempt  to  destroy  the 
complementophile  group  (  =  cytophilic  amboceptoids)  or  to  neutralize 
it  by  means  of  a  suitable  antibody.  The  decision  of  this  question 
must  be  left  to  further  detailed  investigations. 

In  the  course  of  our  experiments  we  met  with  a  very  curious  phe- 
nomenon, one  not  only  of  some  practical  significance,  but  also  of 
considerable  theoretical  interest.  Our  experiment  showed  exactly 
the  opposite  behavior  which  Bordet  had  found.  That  is  to  say,  where 
Eordet  found  that  the  antiserum  acts  as  an  antiamboceptor  on  the 
amboceptor  anchored  to  the  cell,  and  that  this  action  is  overcome 
by  normal  rabbit  serum,  one  of  our  cases  represents  the  reverse  of 
this.  We  see,  therefore,  that  it  can  happen  that  the  antiamboceptor 
as  such  does  not  act,  but  requires  the  addition  of  normal  rabbit  serum 
before  exerting  its  action.  We  have  constantly  observed  that  in  a 
"curative"  experiment,  i.e.,  after  a  previous  binding  of  amboceptor 
and  cell,  large  amounts  of  the  antiserum  produced  by  means  of  im- 
mune serum  were  unable  to  prevent  haemolysis.  The  following  proto- 
col may  serve  as  an  example: 

To  each  of  a  series  of  test-tubes,  containing  decreasing  amounts 
of  the  antiserum,  1  cc.  of  ox  blood  was  added.  This  blood,  after 
having  previously  been  sensitized  with  0.003  cc.  (  =  1J  amboceptor 
units)  of  an  amboceptor  obtained  from  a  rabbit  by  immunization 
with  .ox  blood,  was  freed  from  serum  constituents  by  centrifuging 
and  then  used  in  the  test.  After  digesting  the  mixtures  for  half  an 
hour  the  blood-cells  were  centrifuged  off  and  the  sediments,  to 
which  0.1  cc.  guinea-pig  serum  was  added  as  complement,  were 


568 


COLLECTED  STUDIES  IN    IMMUNITY, 


suspended  in  salt  solution.     The  result  of  the  experiment  is  shown 
in  the  following  table: 

TABLE    I. 


Amount  of  the  Antiserum 

(derived  from  a  goat  by 

treatment  with  an  am- 

boceptor,  the  result  of 

Amount  of  Haemolysis. 

immunizing  a  rabbit 

with  ox  blood) 

cc. 

0.1 

complete 

0.05 

well-marked 

0.025 

moderate 

0.015 

little 

0.01 

0 

0.005 

faint  trace 

0.0025 

very  little 

0.0015 

moderate 

0.001 

almost  complete 

0.0005 
0.00025 

complete 
complete 

0 

complete 

Here  we  see  the  curious  result  that  with  a  certain  excess  of  the 
antiserum  there  is  no  inhibition  of  haemolysis.  This  paradoxical 
phenomenon  we  observed  only  with  the  antiserum  produced  by 
immune  serum  injections,  and  then  only  in  the  "curative"  experi- 
ment. If  the  antiserum  was  used  for  "protective"  experiments, 
i.e.,  mixed  with  amboceptor  previous  to  adding  the  blood-cells,  or 
if  the  antiserum  produced  by  injections  of  normal  serum  was  employed, 
the  course  of  the  experiment  was  entirely  uniform,  an  increase  in  the 
amount  of  antiserum  causing  an  increase  in  the  antilytic  action. 
For  the  present  we  are  unable  to  say  whether  we  are  here  dealing 
with  an  essential  difference  between  the  antiserum  produced  by 
normal  serum  and  that  produced  by  immune  serum,  or  whether  we 
have  to  do  with  an  individual  fluctuation.  So  far  as  the  mechanism 
of  the  phenomenon  is  concerned  we  were  able  to  clear  up  at  least 
one  point,  namely,  that  the  essential  factor  in  the  experiment  is  the 
presence  or  absence  of  the  very  small  quantities  of  normal  rabbit 
serum  which  contains  the  amboceptor.  Thus  if  the  blood-cells  are 
sensitized  with  amboceptor  without  subsequently  removing  the  serum 
by  centrifuging,  it  will  be  found  that  the  course  of  the  "curative" 
experiment  is  perfectly  regular.  There  is  no  inhibition  of  the  antilytic 
action  with  an  excess  of  antiserum.  The  same  holds  true  if  we  sepa- 


MECHANISM   OF  THE  ACTION   OF  ANTIAMBOCEPTORS.      569 

rate  the  sensitized  blood-cells  by  centrifuge  and  replace  the  serum 
fluid  with  the  corresponding  amount  of  normal  serum  (in  our  cases 
0.003  cc.).  The  active  substance  contained  in  normal  serum  is 
thermostable  at  56°  C.;  but  is  destroyed  by  heating  for  half  an  hour 
to  100°  C.  The  following  experiment  may  serve  as  an  illustration: 

The  blood-cells  which  have  been  sensitized  with  0.003  cc.  serum 
and  then  separated  by  centrifuge  are  treated  with  a  considerable 
excess  (0.5  cc.)  of  the  antiserum.  This  amount  corresponds  to  that 
quantity  which  by  itself  is  just  able  to  overcome  the  antilytic  action. 
To  this  mixture  are  added  decreasing  amounts  of  normal  rabbit  serum 
which  has  been  heated  to  56°  C.  and  to  100°  C.  After  allowing  the 
mixture  to  stand  for  half  an  hour  the  blood-cells  are  centrifuged 
off  and  suspended  in  salt  solution  to  which  0.1  cc.  guinea-pig  serum 
(complement)  is  added. 

The  result  is  shown  in  the  following  table: 


TABLE    II. 


Amount  of  Normal  Rabbit 
Serum. 

1  cc.  5%  Ox  Blood  (sensitized  with  0.003  cc.)  +  0.5  cc. 
Antiserum+  Normal  Rabbit  Serum. 

a. 
Heated  to  56°  C. 
Amount  of  Haemolysis. 

6. 
Heated  to  100°  C. 
Amount  of  Haemolysis. 

0.005 
0.003 
0.0015 
0.001 
0.0005 
0 

0 
0 

little 
moderate 
complete 
complete 

complete 

This  shows  us  what  a  tremendous  effect  the  presence  or  absence 
of  a  small  amount  of  normal  serum  can  exercise.  This  of  course 
at  once  explains  the  difference  which  manifests  itself  between  the 
"curative"  and  the  "protective"  experiments.  In  the  latter,  it  will 
be  recalled,  the  amboceptor  and  antiamboceptor  are  first  mixed. 
All  of  the  normal  serum  constituents,  therefore,  come  into  action; 
whereas  in  the  "curative"  experiment  these  are  removed  when  the 
blood-cells  are  centrifuged. 

How  are  we  to  conceive  the  mechanism  of  this  action?  Phe- 
nomena in  which  an  excess  of  a  certain  substance  produces  a 
change  in  the  character  of  the  reaction  are  frequently  due  to  the 


570  COLLECTED  STUDIES  IN  IMMUNITY. 

presence  of  other  substances  with  different  properties.  In  the  case 
described  above  there  is  an  absence  of  antilytic  action  with  a  certain 
excess  of  the  antiserum.  If  we  look  at  the  subject  from  this  stand- 
point, we  shall  have  to  assume  that  the  antiserum  contains  two  sub- 
stances,1 one  of  which,  of  course,  is  the  effective  antiamboceptor. 
The  other  substance  would  then  be  the  cause  of  the  inhibition  of 
the  antiamboceptor  action.  Furthermore,  since  this  inhibition  is 
only  brought  about  by  large  quantities  of  the  serum,  this  substance 
would  be  present  in  the  serum  in  much  smaller  amounts  than  the 
former.  The  simplest  explanation  of  the  action  of  this  substance 
seems  to  be  somewhat  as  follows:  We  must  assume  that  this  sub- 
stance's point  of  attachment  is  a  complementophilic  auxiliary  group 
in  the  amboceptor.  The  occupation  of  this  group  so  affects  the 
amboceptor  molecule  that  the  simultaneous  presence  of  antiambo- 
ceptor no  longer  prevents  the  combination  with  complement.  Such 
a  behavior  would  be  analogous  to  an  observation  published  by  Ehr- 
lich  and  Marshall.2  At  that  time,  by  means  of  a  differentiating 
method  made  available  for  one  particular  instance 3  by  Marshall 
and  Morgenroth,  it  was  shown  that  the  amboceptor  anchored  to 
the  cell,  although  it  could  deprive  native  guinea-pig  serum  of  all  its 
complement  functions,  was  unable  to  absorb  the  non-dominant 
complements  if  the  dominant  complement  had  first  been  neutralized 
by  the  partial  anticomplements  of  Marshall  and  Morgenroth.  In 
other  words,  an  anchoring  of  the  non-dominant  complements  was 
only  possible  after  the  corresponding  complementophile  group 
of  the  amboceptor  had  combined  with  the  dominant  complement. 
In  our  case  we  would  be  dealing  with  an  influence  entirely  similar 
in  principle,  except  that  here  the  influence  is  reversed,  i.e.,  the  affinity 
of  the  amboceptor  to  the  antiamboceptor  is  reduced  by  the  occupa- 
tion of  the  auxiliary  group.  We  believe  that  we  can  show  directly 
that  the  antiamboceptor  is  bound  in  either  case,  but  that  where  .the 
auxiliary  group  is  occupied,  the  union  of  amboceptor  and  antiambo- 

1  We  can  of  course  assume  a  priori  that  an  antiamboceptor  serum  directed 
against   the   complementophile   groups   will   possess   a   multiplicity   of   partial 
antiamboceptors,  for  the  amboceptors  which  take  part  in  the  immunization 
possess   a   large   number   of  different   complementophile    groups,  and   against 
each  of  these  a  particular  antibody  is  conceivable. 

2  Ehrlich  and  Marshall,  1.  c. 

3  H.  T.   Marshall   and  J.   Morgenroth,   Uber  Differenzierung  von    Comple- 
menten  durch  ein  Partialanticomplement.      Centralblatt  f.  Bact.  1902,  Vol.  31, 
No.  12. 


MECHANISM  OF  THE  ACTIOX*  OF  ANTIAMBOCEPTORS.      571 

ceptor  remains  a  loose  one,  while  in  the  other  case  it  becomes  firm. 
The  following  diagram  may  help  to  make  this  clear.     See  Fig.  1. 

We  shall  designate  the  two  complementophile  groups  of  the  ambo- 
ceptor  as  a  and  /?;  the  effective  antiamboceptor  corresponding  to 
group  a  is  a,  the  antibody  fitting  group  /?  is  6.  In  small  quan- 
tities of  antiserum,  b  can  practically  be  disregarded  owing  to  its 
slight  concentration ;  a  therefore  by  occupying  a  prevents  the  comple- 
ment uniting  with  the  amboceptor.  In  larger  quantities  of  anti- 
serum,  however,  b  comes  into  play,  so  that  the  occupation  of  group  /? 


Loose  Union 


FIG  1. — a  and  /?:  Complementophile  groups  of  the  amboceptor.  a  and  6  are 
Partial  Substances  of  the  Antiserum.  a  is  the  effective  Antiamboceptor; 
b  is  the  antibody  which  inhibits  the  action  of  the  antiamboceptor.  c  is  the 
Complement. 

changes  the  reactive  capacity  of  group  a  in  such  a  way  that  either  a 
is  not  bound  at  all  wrhile  the  corresponding  complement  is,  or  so 
that,  wh  le  a  may  still  be  bound,  the  union  is  such  a  loose  one  that 
the  complement  still  has  access.  We  shall  see  that  the  latter  pos- 
sibility is  the  more  probable.  First,  however,  it  will  be  necessary 
for  us  to  understand  clearly  the  manner  in  which  normal  rabbit 
serum  overcomes  the  influence  of  the  antiserum  constituent  6.  In 
view  of  what  has  been  said  this  will  not  be  difficult,  for  it  is  but  a 


572 


COLLECTED  STUDIES  IN   IMMUNITY. 


natural  consequence  for  us  to  assume  that  normal  rabbit  serum  con- 
tains the  corresponding  counter-group  /?  in  such  high  concentration 
that  even  small  amounts  are  able  to  neutralize  b  and  so  prevent  its 
union  with  the  amboceptor  anchored  by  the  cell.  See  Fig.  2. 

Coming  now  to  the  question  whether,  after  group  /?  is  occupied, 
group  a  no  longer  reacts  with  a,  or  whether,  while  the  reaction  takes 
place,  the  union  remains  a  very  loose  one,  we  decided  this  according 
to  the  following  considerations.  If  the  latter  assumption  were  cor- 
rect, it  would  follow  that  the  loose  union  should  subsequently  become 


>^^ 


FIG.  2. — /?:  Complementophile  group  of  an  amboceptor  of  normal  serum. 
Otherwise  as  in  Fig.  1. 

firm  if  in  some  way  group  b  could  again  be  freed  from  its  combination 
with  /?.  In  that  case,  evidently,  the  "curative"  action  of  the  anti- 
amboceptor  a  should  become  manifest.  If,  on  the  contrary,  a  has  not 
been  bound  at  all,  this  "curative"  action  should  fail  to  appear  on 
the  removal  of  b. 

Owing  to  the  presence  of  group  /?  in  small  amounts  in  normal 
rabbit  serum  the  possibility  is  given  of  abstracting  the  antigroup  b 
already  bound  to  the  sensitized  cell.  We  have  at  once  taken  advan- 
tage of  this  fact,  and  attacked  the  question  experimentally  as  follows: 

Sensitized  blood-cells  are  digested  with  an  excess  of  the  antiserum 


MECHANISM  OF  THE  ACTION  OF  ANTIAMBOCEPTORS.      573 


(0.25  cc.).  After  centrifuging,  decreasing  amounts  of  inactivated 
normal  rabbit  serum  are  added  to  the  sediments,  and  the  mixtures 
again  centrifuged.  The  blood-cells  thus  separated  are  suspended 
in  0.1  cc.  salt  solution  containing  0.1  cc.  guinea-pig  serum.  The  result 
is  shown  in  the  following  table: 

TABLE    III. 


In  active  Normal  Rabbit 

Serum. 

Amount  of  Haemolysis. 

cc. 

0.01 

1 

0.006 
0.003 

I  little  to  moderate 

0.0015 

J 

0 

complete 

This  table,  therefore,  shows  that  sensitized  blood-cells  which  have 
been  treated  with  an  excess  of  antiamboceptor  and  then  freed  from 
all  free  serum  constituents  by  centrifuging  can  be  deprived  of  a  con- 
siderable portion  1  of  the  antiserum  constituent  b  by  subsequently 
digesting  them  with  small  amounts  of  normal  rabbit  serum,  thus 
again  allowing  the  antiamboceptor  action  to  become  manifest.  It 
is  permissible,  therefore,  to  assume  that  the  antiamboceptor  a  had 
been  bound  and  that  the  union  had  remained  a  loose  one  owing  to 
the  occupation  of  group  /?,  Owing  to  the  looseness  of  the  union  a 
and  a  the  complement  was  not  prevented  from  combining  with  the 
amboceptor. 

We  have  gone  into  the  analysis  of  this  case  with  such  detail  because 
it  again  shows  how  complicated  is  the  mechanism  of  amboceptors 
and  yet  how  easy  it  is  by  means  of  the  amboceptor  theory  to  bring 
these  apparently  paradoxical  phenomena  into  harmony.  In  this  case 
we  are  certainly  dealing  with  extraordinarily  complex  conditions, 
conditions  in  which  Bordet's  rudimentary  sensitization  theory  is 
entirely  helpless. 

The  phenomenon  just  described  possesses  a  certain  practical 
significance  in  so  far  as  it  could  easily  lead  to  the  erroneous  assump- 

1  It  is  likely  that  the  reason  why  the  inhibiting  action  cannot  be  entirely 
brought  out  by  this  means  is  that  the  union  of  6,  once  it  is  bound,  rapidly  be- 
comes firm,  thus  permitting  only  a  partial  dissolution  by  means  of  free  ft.  In 
any  event  this  experiment  clearly  exhibits,  as  already  stated,  exactly  the  re- 
verse behavior  of  that  shown  by  Bordet's. 


574  COLLECTED  STUDIES  IN  IMMUNITY 

tion  that  the  antiamboceptor  acts  only  in  "protective"  experiments,, 
but  is  unable  to  act  on  amboceptor  already  anchored  by  the  blood- 
cells.  In  order  to  orientate  ourselves  concerning  this  last  question,  we 
would  of  course  begin  by  using  an  excess  of  antiamboceptor,  expecting 
very  naturally,  if  the  antiamboceptor  exerts  any  influence  whatever 
on  the  anchored  amboceptor,  that  this  influence  will  most  likely 
become  manifest  with  large  amounts  of  antiamboceptor.  Further- 
more, it  can  then  happen  that  the  conditions  obtaining  are  those  of 
the  zone  in  which  the  curative  action  obtained  with  smaller  doses 
is  concealed,  owing  to  the  excess  of  antiamboceptor.  This  may 
perhaps  account  for  Morgenroth's  negative  findings;1  the  antiambo- 
ceptor serum  employed  by  us  was  also  used  by  that  author. 

The  demonstration  of  the  fact  that  the  antiamboceptors  pro- 
duced by  immunization  are  usually  directed  against  the  complemento- 
phile  groups  calls  for  a  correction  of  certain  deductions  based  on 
our  earlier  conception  of  antiamboceptors  as  being  directed  against 
the  cytophile  group.  We  must  therefore  concede  that  Bordet  is 
correct  when  he  refuses  to  accept  our  method  of  differentiating  partial 
amboceptors  by  means  of  antiamboceptors,  a  method  which  we  pub- 
lished in  the  Sixth  Communication  on  Hsemolysins.2  Our  experi- 
ments at  that  time  dealt  with  an  amboceptor  of  an  immune  serum 
derived  from  a  rabbit  by  treatment  with  ox  blood.  This  amboceptor 
could  be  complemented  either  with  guinea-pig  serum  or  goat  serum.  In 
complementing  with  goat  serum  so  much  more  amboceptor  is  necessary 
that  the  absence  of  the  antiamboceptors'  action  must  be  ascribed  to 
the  antiantilytic  action  of  the  normal  amboceptors  present.  But 
this  correction  does  not  signify  that  the  conclusion  as  to  the  plurality 
of  the  amboceptors  must  be  abandoned.  On  the  contrary  this  con- 
clusion is  confirmed  by  so  many  weighty  arguments  of  a  different  kind 
that  the  existence  of  partial  amboceptors  must  now  be  classed  as  one  of 
the  facts  in  immunity.  We  need  only  call  attention  to  a  point  con- 
tained in  our  Sixth  Communication,  namely,  that  by  mutual  elective 
absorption  we  have  shown  that  immunization  of  animals  with  ox 
blood  results  in  the  formation  of  two  fractions  of  amboceptors,  one 
of  which  acts  only  on  ox  blood,  the  other  also  on  goat  blood;  and 
that  immunization  with  goat  blood  has  exactly  analogous  reverse 


1  J.  Morgenroth,  Deflection  of  Complement  by  Means  of  Haemolytic  Ambo- 
eeptors.     Centralblatt  Bact.  1904,  Vol.  35,  No.  4. 
2Ehrlich  and  Morgenroth.     See  page  88. 


MECHANISM  OF  THE  ACTION   OF  ANTIAMBOCEPTORS.      575 

results.  The  plurality  of  amboceptors  is  further  demonstrated  by 
the  results  of  the  isolysin  experiments  published  by  Ehrlich  and 
Morgenroth,1  for  in  these  experiments  the  presence  of  antibodies 
acting  against  the  complementophile  group  of  the  amboceptor  can 
be  excluded.  The  fact  that  we  have  drawn  an  incorrect  conclusion 
from  one  single  experiment  certainly  does  not  justify  Bordet  in  deny- 
ing the  existence  of  a  plurality  of  antibodies  (especially  amboceptors) 
in  a  given  immune  serum;  the  correctness  of  our  view  is  established 
by  a  number  of  incontestable  experiments. 

Bordet's  arguments  concerning  deflection  of  complement  by  an 
excess  of  amboceptor  may  be  answered  in  the  same  manner.  Even 
granted  that  Morgenroth's  view2  is-incorrect,  namely,  that  the  inhibi- 
tion of  haemolysis  on  the  addition  of  an  amboceptor-antiamboceptor 
mixture  is  due  to  a  deflection  of  complement,  this  would  not  in  the 
least  refute  the  results  obtained  by  Xeisser  and  Wechsberg  with 
bactericidal  sera.  In  these  experiments  absolutely  no  antiambo- 
ceptor  is  present ;  there  are  merely  bacteria,  amboceptor,  and  comple- 
ment. Despite  this,  however,  there  is  no  bactericidal  action  when  a 
certain  excess  of  amboceptor  is  present.  The  only  explanation  for 
this  is  the  one  offered  by  Xeisser  and  Wechsberg,3  namely,  that  the 
complement  is  deflected  from  the  amboceptor  combined  with  the 
cells  by  the  free  amboceptor.  This  explanation  has  also  been  accepted 
by  Lipstein,4  who  controverted  a  number  of  objections  which  had 
been  made  by  various  authors.  Bordet  does  not  even  attempt  to 
controvert  our  explanation,  but  contents  himself  by  saying:  "Pour 
nous,  la  theorie  de  la  deviation  du  complement  par  Pambocepteur 
est  une  legende."  Needless  to  say  this  will  have  little  effect  on  our 
view. 

It  is  thus  seen  that  Bordet's  recent  experiments  have  furnished 
additional  important  confirmation  of  the  amboceptor  theory.  Analysis 
of  the  antiamboceptor  action  clearly  demonstrates  the  fact  that  the 
amboceptor  possesses  other  affinities  besides  those  of  the  cytophile 
group;  and  the  circumstance  that  the  occupation  of  these  groups 
bars  the  action  of  the  complement  shows  that  they  are  complemento- 
phile in  character.  Bordet's  attack  on  the  receptor  theory  has  thus 

1  Ehrlich  and  Morgenroth,  Third  Communication.     See  page  23. 

2  J.  Morgenroth,  1.  c. 

3  M.  Neisser  and  Wechsberg.     See  page  120. 

4  A.  Lipstein,  Centralblatt  fur  Bacteriologie,  1902,  Vol.  31,  No.  10;   see  also 
page  132  of  this  volume. 


576  COLLECTED  STUDIES  IN  IMMUNITY. 

failed  utterly;   his  experiments,  on  the  contrary,  are  to  be  welcomed 
as  supplementing  the  arguments  supporting  the  amboceptor  theory.1 

1  The  mistake  contained  in  our  previous  conception  of  antiamboceptors, 
that  they  were  antibodies  directed  against  the  cytophile  group,  is  essentially 
one  regarding  the  situation  of  the  point  of  attack.  In  this  connection  we  may 
look  upon  certain  chemical  substitutions  as  furnishing  ready  comparison;  for 
example,  the  different  substances  resulting  when  the  benzole  nucleus  is  substi- 
tuted in  the  ortho,  meta,  or  para  positions.  Considering  how  difficult  these 
problems  are,  it  is  not  surprising  that  a  statement  concerning  localization  will 
now  and  then  be  made  which  subsequent  deeper  study  shows  must  be  corrected. 
Even  so  high  an  authority  as  Kekule  once  erred  in  denning  a  compound,  and 
yet  this  did  not  in  the  least  affect  his  fruitful  hypothesis.  In  our  case  after  the 
way  had  been  cleared  by  the  demonstration  of  the  "blocking  of  complements" 
(the  nature  of  which  corresponds  to  an  antiamboceptor  action),  and  by  the 
studies  of  Pfeiffer  and  Friedberger,  it  was  an  easy  matter  to  arrive  at  a  correct 
interpretation  and  transfer  the  site  of  the  antiambocepter's  action  to  the  comple- 
mentophile  group.  It  is  at  once  clear  that  this  merely  fulfills  an  old  postulate 
of  the  side-chain  theory.  It  would  therefore  be  interesting  to  see  how  Bordet 
could  explain  the  facts  according  to  his  sensitization  theory,  and  to  have  him 
show  how  the  sensitizers,  which  he  believes  do  not  combine  with  the  comple- 
ment, excite  the  production  of  substances  whose  constitution  is  just  what  would 
be  demanded  of  immunization  products  of  "complementophile  groups." 


XLI.   A    GENERAL    REVIEW  OF  THE   RECENT   WORK 

IN  IMMUNITY.1 

By  PAUL  EHRLICH. 

Two  years  have  elapsed  since  the  appearance  of  my  "Collected 
Studies  in  Immunity"  in  Germany,  and  now  that  the  book  is  about 
to  appear  on  the  other  side  of  the  ocean  it  is  a  pleasure  for  me  to 
review  briefly  the  progress  made  in  that  time,  naturally  without 
pretending  to  give  a  complete  resume  of  the  literature. 

I  may  at  once  say,  however,  that  very  little  really  new  has  been 
added  to  the  views  formulated  by  myself  and  my  collaborators,  and 
that  the  stereochemical  conception  of  the  immunity  reaction,  despite 
numerous  attacks,  has  proven  itself  able  to  dominate  every  phase  of 
the  subject. 

The  arithmetical  view  of  the  toxin-antitoxin  reactions  and  their 
analogues,  which  was  introduced  chiefly  by  Arrhenius  and  Madsen, 
has  invariably  shown  itself  to  be  untenable.  It  has  led  to  a  numer- 
ical science  which  is  far  removed  from  the  principles  of  biological 
investigations  and  from  the  experimental  results  underlying  these. 
On  the  other  hand,  so  able  an  authority  as  Nernst  at  once  recognized 
that  the  laws  of  chemical  equilibrium  are  not  applicable  to  mixtures 
of  toxin  and  antitoxin.  In  addition  to  this  von  Dungern,  Morgen- 
roth,  and  Sachs  have  collected  considerable  new  experimental  evi- 
dence which  demonstrates  absolutely  that  the  toxin-antitoxin 
combination  gradually  becomes  firm,  although  it  may  in  some 
instances  be  quite  loose  in  the  first  stage.  The  complex  constitution 
of  the  poison  solutions  has  thus  been  conclusively  demonstrated; 
and  I  may  also  remind  the  reader  that  there  can  also  no  longer  be 
any  question  as  to  the  independent  existence  of  toxons  in  diphtheria 
poison,  for  van  Calcar  has  succeeded  in  a  direct  separation  of  these 
bodies.2 

1  This  chapter  is  written  expressly  for  this  American  edition. 

2  van  Calcar  effected  this  by  means  of  an  ingenious  dialyzing  procedure 
(Berlin,  klin.  Wochenschr.  No.  39,  1904).     Certain  objections  raised  by  Romer 

577 


578  COLLECTED  STUDIES  IN   IMMUNITY. 

In  view  of  the  extraordinary  success  which  physical  chemistry 
has  scored,  it  is  readily  understood  how  tempting  it  was  for  so  emi- 
nent a  representative  of  this  science  as  Arrhenius  to  apply  its  princi- 
ples to  the  new  field  of  immunity.  I  have  always  emphasized  the 
chemical  nature  of  the  reaction,  and  am  glad  therefore  that  the 
attempt  to  apply  these  principles  has  been  made.  It  has  demon- 
strated anew  that  the  phenomena  of  animate  nature  represent  merely 
the  resultants  of  infinitely  complex  and  variable  actions,  and  that 
they  differ  herein  from  the  exact  sciences,  whose  problems  can  be- 
treated  mathematically.  The  formulas  devised  by  Arrhenius  and 
Madsen  for  the  reaction  of  toxins  and  antitoxins  explain  absolutely 
nothing.  Even  in  particularly  favorable  cases  they  can  merely 
represent  certain  experimental  results  in  the  form  of  interpolation 
formulas.  Neither  do  I  believe  that  the  phenomena  observed  in 
toxins  and  antitoxins  bear  any  relation  to  the  processes  of  colloid 
chemistry.  The  attempt  which  has  been  made  to  interpret  the 
immunity  reaction  from  the  standpoint  of  colloid  chemistry,  a  sub- 
ject itself  more  or  less  obscure,  is  based  on  purely  external  analogies. 
I  see  absolutely  no  advantage  in  such  a  method,  and  I  have  grave 
fears  that  it  will  result  in  checking  further  progress  along  this  line. 
Structural  chemistry,  on  the  other  hand,  has  not  only  served  to 
explain  all  the  phenomena  in  immunity  studies,  but  has  also  proved 
a  valuable  guide  in  indicating  the  lines  along  which  further  progress 
might  be  made.  The  limitations  of  colloid  chemistry  have  already 
manifested  themselves,  and  enthusiastic  advocates  of  this  science 
have  been  compelled  to  assume  the  existence  of  specific  atomic 
groupings  in  accordance  with  my  views.  I  therefore  see  no  reason 
for  abandoning  the  views  expressed  in  my  receptor  theory,  a  theory 
in  complete  accord  with  the  principles  of  synthetic  chemistry.  My 
decision  finds  additional  support  in  the  fact  that  the  studies  in 
immunity  are  constantly  bringing  to  light  new  observations  best 
harmonized  with  the  views  of  structural  chemistry.  Thus  I  may 
remind  the  reader  that  Morgenroth  has  recently  very  cleverly  proved 
the  postulate  that  the  components  of  the  neutral  toxin-antitoxin 
combination  can  be  restored.  This  author  succeeded  in  completely 
recovering  the  two  components  of  a  neutral  mixture  of  cobra  venom 

(Berl.  klin.  Wochenschr.  No.  8,  1905)  have  been  effectually  answered  by  van 
Calcar  by  means  of  some  additional  experiments,  and  by  the  demonstration 
that  the  membranes  employed  by  Homer  were  unsuitable  (Berl.  klin.  Woch. 
No.  43,  1905). 


A  GENERAL  REVIEW  OF  THE  RECENT  WORK  IN  IMMUNITY.  579 

and  antitoxin  by  means  of  an  ingenious  method.  But  even  here  we 
are  not  dealing  with  a  reversible  reaction,  for  it  requires  certain 
manipulations  to  disrupt  the  neutral  combination;  thus,  in  the  case 
of  cobra  venom,  the  addition  of  hydrochloric  acid  is  necessary.  The 
neutral  cobra-venom-antitoxin  combination  therefore  behaves  like  a 
glucoside,  which  in  itself  is  entirely  stable,  but  is  split  up  by  the  addi- 
tion of  hydrochloric  acid. 

Besides  this,  the  interesting  investigations  recently  published  by 
Obermayer  and  Pick,1  on  the  production  of  immune  precipitins  by 
means  of  chemically  altered  albuminous  bodies,  are  of  particular  sig- 
nificance in  connection  with  the  chemical  conception  of  the  immunity 
reaction.  These  authors  succeeded,  by  iodizing,  nitrifying,  and 
diazotizing  animal  albuminous  bodies,  in  so  changing  them  that, 
when  introduced  into  the  organism  of  the  same  or  of  different  species, 
they  excited  the  production  of  precipitins  which  lacked  specificity. 
These  precipitins,  however,  were  strictly  specific  for  their  respective 
iodized  albumins,  xanthoproteids,  or  diazo-albumins,  no  matter  from 
what  animal  species  the  albumins  were  derived. 

We  see,  therefore,  that  the  introduction  of  a  certain  chemical  group 
into  the  albumin  molecule  completely  alters  the  latter's  power  to 
excite  the  production  of  antibodies.  This  certainly  corresponds 
entirely  to  the  view  that  the  production  of  antibodies  is  dependent 
on  the  chemical  constitution  of  the  exciting  agent,  a  view  which  finds 
expression  in  my  receptor  theory. 

The  heuristic  value  of  the  receptor  idea,  the  idea  which  underlies 
my  side-chain  theory,  can  best  be  appreciated  by  studying  the  devel- 
opment of  our  knowledge  concerning  the  cy  to  toxins  of  blood  serum. 
As  a  prototype  of  these  substances  the  hsemolysins  occupy  a  promi- 
nent place  in  this  volume.  The  view  that  the  haemolytic  immune 
bodies  are  amboceptors  has  been  proven  to  be  correct  in  every  case, 
thus  conclusively  showing  that  Bordet's  sensitization  theory  is  un- 
tenable. To  begin,  the  observations  of  M.  Neisser  and  Wechsberg, 
that  the  action  of  bactericidal  sera  depends  not  only  on  the  absolute 
but  on  the  relative  concentration  of  amboceptor  and  complement, 
presented  conditions  which  could  not  be  harmonized  with  Bordet's 
views.  On  the  other  hand,  they  were  readily  explained  in  accord- 
ance with  the  side-chain  theory  by  assuming  that  the  complement 
was  deflected  by  an  excess  of  amboceptor.  But  even  if  this  expla- 

1  Centralbl.  f.  Physiologie,  Vol.  XIX,  No.  23. 


580  COLLECTED  STUDIES  IN  IMMUNITY. 

nation  is  not  the  correct  one;  as  Gay  has  recently  stated,  it  would  in 
no  way  affect  the  soundness  of  the  amboceptor  theory.  The  exist- 
ence of  amboceptors  is  confirmed  by  so  many  experimental  consider- 
ations that  it  is  no  longer  a  postulate  of  the  theory,  but  is  practically 
the  direct  expression  of  observed  phenomena.  The  term  amboceptor, 
of  course,  is  used  merely  to  express  the  two-sided  affinity,  to  the 
cell  on  the  one  hand  and  to  the  complement  on  the  other.  The 
affinity  of  the  amboceptor  to  the  cell  was  demonstrated  by  the  com- 
bining experiments  published  by  Morgenroth  and  myself;  and  the 
direct  union  of  amboceptor  and  complement  is  confirmed  by  a  host 
of  decisive  observations.  Of  these,  it  will  suffice  to  mention  the 
test-tube  demonstration  of  complementoids  which  occupy  the  com- 
plementophile  groups  of  the  amboceptor.  This  demonstration  has 
since  been  effected  in  other  ways  (Fuhrmann,  Muir,  Browning,  and 
Gay),  so  that  the  existence  of  complementoids  is  no  longer  evidenced 
merely  by  the  possibility  of  producing  anticomplements  by  means  of 
inactivated  serum,  but  is  demonstrated  primarily  by  the  unmistak- 
able interference  of  the  complementoids  in  hsemolytic  test-tube 
experiments.  It  is  not  necessary  that  complementoids  should  always 
exert  an  inhibiting  action  on  haemolysis;  for  it  is  obvious  that  changes 
in  affinity  may  occur  in  consequence  of  external  influences,  physical, 
chemical,  or  chronological  in  nature.  I  believe  that  changes  in  affinity, 
either  positively  or  negatively,  are  of  the  highest  importance  in  cor- 
rectly understanding  the  course  of  immunity  reactions,  although  I 
do  not  deny  the  influence  of  certain  catalytic  factors  on  these  proc- 
esses (von  Behring,  Morgenroth,  Otto,  and  Sachs).  However,  no 
general  rule  can  be  laid  down.  Experiments  are  constantly  bringing 
forth  surprises,  but  by  diligent  empiricism  it  is  usually  possible  to 
bring  the  many  different  observations  into  harmony  with  a  single 
point  of  view. 

The  original  assumption,  that  amboceptor  and  complement  (at 
least  in  the  case  of  hsemolysins)  exist  free  side  by  side,  and  that  the 
complement  does  not  take  part  in  the  reaction  until  the  amboceptor 
has  been  bound  by  the  cell  (owing  to  an  increase  in  the  affinity  of 
the  complementophile  group), — this  assumption  has  not  proven  ten- 
able in  every  case.  In  addition  to  the  case  described  in  a  previous 
chapter  by  Sachs  and  myself,  we  now  know  of  a  number  of  combi- 
nations, discovered  by  Sachs,  in  which  the  amboceptor  alone  does 
not  unite  with  the  receptor  of  red  blood-cells,  or  does  so  to  only  a 
slight  degree.  By  combining  with  the  complement,  the  amboceptor 


A  GENERAL  REVIEW  OF  THE  RECENT  WORK  IN  IMMUNITY.  581 

has  the  affinity  of  its  cytophile  group  increased,  so  that  now  it  is  able 
to  unite  with  the  cells.  Thus  far,  such  observations  have  been  made 
only  on  normal  amboceptors;  and  this  fact  explains  why  the  numerous 
attempts  of  various  authors  to  separate  normal  haemoly sins,  by  means 
of  absorption  at  low  temperatures,  have  failed.1  The  amboceptors 
obtained  by  immunization,  on  the  other  hand,  regularly  possess  a 
high  affinity  for  the  cell-receptor.  This  is  easily  understood  if  we 
consider  their  mode  of  origin,  for  we  may  perhaps  see  in  this  a  selec- 
tion of  the  groups  with  the  highest  affinity.  Certainly  in  this  case 
the  exception  proves  the  rule;  for  the  mere  fact,  that  in  some  instances 
the  amboceptor  does  not  unite  with  the  cell  until  it  has  first  com- 
bined with  the  complement,  at  once  shows  that  we  cannot  be  dealing 
with  a  sensitization.  On  the  contrary,  this  shows  that  the  ambo- 
ceptor is  an  interbody  in  the  strict  sense  of  the  word.  These  condi- 
tions have  been  most  clearly  brought  out  by  the  experiments  of 
Preston  Kyes  on  cobra  venom.  The  researches  of  Flexner  and 
Noguchi,  as  we  all  know,  showed  that  cobra  venom  by  itself  is  no 
hsemolysin,  but  plays  the  role  of  amboceptor  in  haBmolysis.  The 
most  important  of  the  activators  is  the  one  discovered  by  Kyes, 
namely,  lecithin.  The  relation  between  snake  venom  and  lecithin  is 
really  the  same  as  that  between  amboceptor  and  complement;  but 
the  former  possess  one  great  advantage  for  chemical  analysis, — they 
are  both  stable  substances,  and  thus  contrast  strongly  with  the  highly 
susceptible  substances  found  in  blood  serum.  Hence  what  was 
impossible  in  the  case  of  the  latter  could  readily  be  effected  with 
cobra  venom.  Kyes,  it  will  be  remembered,  has  demonstrated,  ad 
ocular,  the  direct  union  of  cobra  amboceptor  and  lecithin  comple- 
ment, and  has  furthermore  succeeded  in  isolating  the  resulting  com- 
bination, the  cobra-lecithid,  in  pure  form.2 

Thus,  for  the  first  time,  the  conclusion  was  reached  chemically 


1  In  this  connection   I  should  also  like  to  mention  the  interesting  atypical 
behavior  discovered  by  Donath  and  Landsteiner  in  the  amboceptor  reaction. 
These  authors  observed  hsemolytic  autoamboceptors  in  the  serum  of  a  patient 
suffering    from    paroxysmal    ha?moglubinaria.     These    autoamboceptors,    how- 
ever, only  united  with  the  bloods  at  low  temperature. 

2  Kyes  has  recently  continued  his  studies  at  my  laboratory,  and  has  demon- 
strated the  important  fact  that  in  this  formation  of  cobra-lecithid  there  is  a 
true  chemical  synthesis.     The  course  of  this  synthesis  is  such  that  a  fatty  acid 
radical  is  split  off  from  the  lecithin  molecule,  whereupon  the  residual  combina- 
tion, which  corresponds  to  a  monostearyllecithin,  unites  with  the  cobra  ambo- 


582  COLLECTED  STUDIES  IN  IMMUNITY. 

which,  as  a  result  of  biological  experiences,  I  had  always  looked 
forward  to. 

The  correctness  of  the  amboceptor  theory  formulated  by  Morgen- 
roth  and  myself  is  confirmed  by  another  important  link  in  the  chain 
of  evidence.  As  far  back  as  1900,  in  the  Croonian  lecture,  I  stated 
that,  according  to  the  amboceptor  theory,  three  antilytic  antibodies 
were  possible.  In  addition  to  the  substances  which  act  as  anticom- 
plements,  we  could  conceive  of  antiamboceptors  of  two  different 
kinds.  One  of  these  inhibits  the  action  of  the  amboceptor  by  pre- 
venting the  union  of  amboceptor  and  cell,  the  other  by  occupying  the 
complementophile  groups.  So  far  as  the  confirmation  of  the  ambo- 
ceptor theory  is  concerned,  it  is  evident  that  the  demonstration  of 
antiamboceptors  directed  against  the  complementophile  group  is  by 
far  the  most  important;  for,  owing  to  the  mode  of  origin,  the  devel- 
opment of  cytophile  groups  of  the  amboceptor  as  reaction  products 
of  the  specific  counter-group  (the  cell-receptor)  is  self-evident.  It 
was  therefore  particularly  gratifying  when  I  found  that  Bordet 
had  recently  furnished  the  demonstration  that  the  antiamboceptor 
developed  with  an  immune,  or  with  a  normal  serum,  is  usually  directed 
against  the  complementophile  group.  This  discovery  very  prettily 
demonstrates  that  the  mechanism  of  hsemolysin  action  proceeds 
according  to  the  amboceptor  theory.  The  error  contained  in  our 
earlier  conception,  that  anti-immune  bodies  were  usually  antibodies 
directed  against  the  cytophile  group,  is  practically  only  an  error  in 
the  localization  of  the  point  of  attack.  This  must  now  be  corrected 
by  regarding  the  complementophile  group  as  the  point  attacked  by 
the  antiamboceptor. 

We  know  that  it  is  possible  to  produce  antiamboceptors  by  im- 
munizing with  normal  serum,  and  Pfeiffer  and  Friedberger  have 
shown  that  the  action  of  the  antiamboceptor  serum  extends  to  all 
the  amboceptors  of  the  animal  species  whose  serum  was  used  for 
immunization.  These  facts  are  only  apparently  a  contradiction  of 
the  specificity  of  amboceptors,  for  the  specificity  of  the  amboceptors 
applies  only  to  the  cytophile  group.  On  the  other  hand,  we  must 
assume  that  all  the  amboceptors  of  the  same  animal  species  are  at 
least  partly  similar  in  structure  so  far  as  the  complementophile 

ceptor.  This  of  course  destroys  the  foundations  of  Noguchi's  calculations,  which 
are  based  on  the  assumption  that  the  reaction  is  reversible;  it  also  disposes  of 
certain  statements  made  by  Bredig. 


A  GENERAL  REVIEW  OF  THE  RECENT  WORK  IN  IMMUNITY.  583 

apparatus  is  concerned.  In  a  way,  therefore,  the  amboceptor  bears 
the  stamp  of  the  animal  species  from  which  it  is  derived.  In  this 
connection  I  have  already  expressed  my  views  in  the  article  entitled 
"  The  Mechanism  of  the  Amboceptor  Action  and  its  Teleological  Sig- 
nificance "  (Koch  Festschrift,  1903):  "In  general,  the  specific  ambo- 
ceptors  possess  a  uniform  structure  in  their  complementophile  por- 
tions, whereas  they  differ  to  a  high  degree  in  their  cytophile  groups, 
whose  physiological  function  is  the  absorption  of  foodstuffs." 

The  studies  of  antiamboceptors  have  demonstrated  that  this  con- 
ception is  correct.  We  see,  therefore,  that  the  specificity  of  the  com- 
plementophile group  of  the  amboceptor,  a  specificity  based  on  the 
animal  species,  at  once  leads  to  a  difference  in  the  amboceptors 
obtained  from  different  species  by  means  of  the  same  immunizing 
material.  In  our  Sixth  Communication  on  Haemolysins,  Morgenroth 
and  I  published  certain  experiments  showing  that  by  means  of  an 
antiamboceptor  we  had  been  able  to  demonstrate  the  diversity  of 
the  amboceptors  produced  in  different  animal  species  by  injections 
of  ox-blood.  This  statement  still  holds  good,  and  its  direct  conse- 
quence demands  that  in  the  practical  application  of  bactericidal  sera, 
we  should  mix  immune  sera  derived  from  different  animals. 

In  view  of  Bordet's  observation,  however,  we  shall  have  to  revise 
our  interpretation  in  so  far  as  the  site  of  this  differentiation  is  con- 
cerned; the  difference  is  in  the  complementophile  group  instead  of 
in  the  cytophile  group.  On  the  other  hand,  we  must  abandon  the 
differentiation  of  partial  amboceptors  in  one  and  the  same  serum  by 
means  of  antiamboceptors,  a  differentiation  which  we  proposed  in 
the  study  on  hsemolysins.  It  must  not  be  thought,  however,  that 
the  pluralistic  conception  of  the  amboceptor  apparatus  is  thereby 
overthrown.  This  conception  is  supported  by  so  many  arguments" 
of  a  different  kind  that  the  existence  of  partial  amboceptors  can  be 
classed  as  one  of  the  demonstrated  facts  in  immunity.  I  may  remind 
the  reader  that  by  means  of  mutual  elective  absorption  it  is  possible 
to  differentiate  the  strictly  specific  portion  of  an  immune  serum 
from  the  non-specific  components  which  give  rise  to  the  group  reac- 
tions. By  this  means  the  presence  of  different  amboceptor  fractions 
could  be  demonstrated  in  the  same  immune  serum.  The  observa- 
tions made  by  Morgenroth  and  myself  on  isolysins  also  speak  strongly 
-in  favor  of  a  multiplicity  of  amboceptors.  In  these  the  possible 
presence  of  antibodies  acting  on  the  complementophile  portion  of  the 
amboceptor  is  absolutely  excluded.  Finally,  if  we  glance  at  the  con- 


584  COLLECTED  STUDIES  IN  IMMUNITY. 

ditions  existing  among  bacteria,  we  find  the  so-called  group  reactions 
showing  that  the  receptor  apparatus  and  the  antisera  possess  a  highly 
multiple  constitution.  This  fact,  as  is  well  known,  has  here  been  of 
great  practical  value.  We  see,  therefore,  that  the  plurality  of  the 
amboceptors,  so  far  as  the  cytophile  group  is  concerned,  is  an  assured 
fact;  the  differentiation  by  means  of  antiamboceptors  directed 
against  the  cytophile  group  can  therefore  very  well  be  foregone. 
The  production  of  antiamboceptors  against  the  cytophile  group  seems 
to  encounter  particular  difficulties,  for  the  complementophile  group 
always  finds  the  corresponding  counter  group  in  the  organism  more 
readily  than  does  the  cytophile  group,  and  therefore  is  alone  bound 
by  the  tissue  receptors.  It  is  possible  that  in  order  to  successfully 
immunize  with  cytophile  groups,  it  will  be  necessary  to  isolate  these 
groups.  The  latter  might  be  accomplished  by  neutralizing  the  com- 
plementophile group  with  the  corresponding  antibody,  or  by  destroy- 
ing this  group  (=cytophilic  amboceptoids). 

In  any  event  these  studies  confirm  the  correctness  of  the  ambo- 
ceptor  theory,  i.e.,  that  there  is  a  direct  combination  of  amboceptor 
and  complement.  To  repeat,  therefore,  the  specificity  of  the  ambo- 
ceptors applies: 

(1)  To  the  receptor  employed  in  immunization,  and  this  mani- 
fests itself  in  the  configuration  of  the  haptophore  group ;  and 

(2)  To  the  animal  species  from  which  the  amboceptor  is  derived. 
The  latter  kind  of  specificity  shows  itself  in  the  structure  of  the  com- 
plementophile apparatus,   which,   as  we  know,  consists  of  a  large 
number  of  individual  complementophile  groups.     To  this  plurality 
of  the  complementophile  groups  there  corresponds  a  plurality  of  com- 
plements as  can  hardly  longer  be  questioned.     So  far  as  the  consti- 
tution of  the  complement  is  concerned,  the  fact  that  it  is  made  up  of 
a  haptophore  and  a  toxophore  group  is  sufficiently  proven  by  test- 
tube    experiments.     The    indirect    method    first    employed    for    the 
demonstration  of  the  haptophore  group,  namely,  by  the  production 
of  anticomplements,  can  therefore  be  dispensed  with. 

However,  I  am  convinced  that  just  as  normal  body-fluids  so  often 
contain  anticomplements,  it  will  also  be  found  possible  to  produce 
these  by  immunization.  But  as  Moreschi  has  well  pointed  out,  the 
experiments  by  which  it  was  sought  to  demonstrate  the  production 
of  anticomplements  are  not  absolutely  conclusive.  Recent  studies 
by  Gengou,  Moreschi,  and  Gay  have  shown  that  in  the  immunization 
with  serum,  antibodies  directed  against  the  albuminous  constituents 


A  GENERAL  REVIEW  OF  THE  RECENT  WORK  IN  IMMUNITY.  585 

are  formed  which,  by  uniting  with  the  corresponding  albuminous 
bodies,  possess  the  property  of  exerting  anticomplementary  effects. 
In  this  case,  therefore,  the  anticomplement  action  is  brought  about 
by  the  interaction  of  two  components,  one  present  hi  the  serum  of 
the  immunized  animal  and  the  other  in  the  serum  of  that  animal 
species  whose  serum  was  used  for  immunization  (Moreschi).  It  is 
clear,  of  course,  that  here  the  dissolved  albuminous  substances,  not 
the  complements,  were  the  antigens.  This  being  the  case,  the  demon- 
stration of  anticomplements  produced  by  immunization  becomes 
extremely  difficult,  and  it  must  be  left  for  future  investigations  to 
see  whether  it  is  at  all  possible  to  differentiate  these  substances  from 
those  antibodies  against  albuminous  substances  which  exert  an  anti- 
complement  action.  So  far  as  the  mechanism  of  the  described  anti- 
complement  action  is  concerned,  I  do  not  think  that  the  observations 
of  Moreschi  and  Gay,  that  absorption  of  complement  is  associated  with 
precipitation,  necessarily  mean  that  precipitation  and  anticomplement 
have  any  causal  relationship.  In  fact  it  seems  reasonable  to  assume, 
in  accordance  with  Gengou's  first  explanations,  that  the  property  of 
binding  the  complements  is  exercised  by  the  albuminous  bodies  sen- 
sitized with  the  specific  amboceptor.  We  would  have  to  conceive 
this  somewhat  in  this  fashion,  that  just  as  when  immunizing  with 
cells,  agglutinins  and  amboceptors  are  formed,  so  also  when  immuniz- 
ing with  dissolved  albuminous  bodies  two  kinds  of  antibodies  are 
formed,  precipitins  and  amboceptors.  If  the  latter,  however,  are 
really  amboceptors  in  the  sense  of  Ehrlich  and  Morgenroth,  we  must 
demand  that  they  will  have  the  same  properties  which  we  have  always 
ascribed  to  the  amboceptor  type.  As  a  matter  of  fact,  the  experiment 
shows  that  this  is  the  case.  These  albumin  amboceptors  also,  in  order 
to  react  with  the  complements,  must  have  the  affinity  of  their  com- 
plementophile  apparatus  raised,  only  in  the  present  case  this  is  effected 
by  the  combination  of  the  amboceptor  with  the  susceptible  body,  the 
albumin.  We  see,  therefore,  that  this  anticomplementary  action  cor- 
responds to  the  deflection  of  complement  through  an  excess  of  im- 
mune body,  first  described  by  M.  Neisser  and  Wechsberg.  Only  in 
this  case  the  deflecting  amboceptor  is  of  a  different  kind,  and  needs 
first  to  react  with  the  corresponding  receptor. 

Through  the  researches  of  Wassermann  and  Schiitze  and  of  Uhlen- 
huth,  one  class  of  antibodies  against  dissolved  albumins,  namely,  the 
precipitins,  has  been  used,  as  is  well  known  to  differentiate  albuminous 
bodies  of  various  origin.  These  have  thus  come  to  be  successfully 


586  COLLECTED  STUDIES  IN  IMMUNITY. 

employed  in  the  forensic  demonstration  of  the  origin  of  blood-stains. 
The  same  thing,  of  course,  was  possible  in  the  case  of  the  albumin 
amboceptors. 

This  fact  has  recently  been  taken  advantage  of  by  M.  Neisser  and 
Sachs,1  who  have  devised  a  procedure  by  which,  by  deflecting  hsemo- 
lytic  complements  by  means  of  albuminous  bodies  loaded  with  am- 
boceptor,  they  diagnosticate  human  blood,  etc.  The  study  of  im- 
munity thus  furnishes  two  biological  methods  for  deciding  a  point  of 
vital  importance  in  forensic  medicine,  namely,  the  origin  of  blood- 
stains. Considering  the  extreme  importance  of  tests  of  this  kind,  I 
am  convinced  that  hereafter  it  will  be  well  to  use  this  method  in 
addition  to  the  well-tried  Uhlenhuth-Wassermann  reaction. 

This  brief  resume,  I  believe,  covers  the  chief  points  which  have 
recently  come  up  for  discussion,  and  it  is  indeed  gratifying  to  me  that 
all  the  vital  questions  have  been  decided  in  favor  of  my  views.  I 
have  gladly  applied  the  results  obtained  in  experimental  investiga- 
tions to  an  extension  of  my  views,  for  it  is  obvious,  considering  the 
rudimentary  character  of  a  new  science,  that  any  successful  prosecu- 
tion of  the  work  will  also  extend  the  theoretical  conceptions.  If  then, 
in  spite  of  this,  all  the  facts  brought  to  light  fit  naturally  into  the 
views  formulated  by  me,  I  regard  this  as  additional  evidence  that 
these  views  are  not  so  much  a  theory  as  a  necessary  abstraction  of  the 
observed  facts,  an  abstraction  which  is  necessary  not  only  in  order  to 
obtain  a  clear  and  harmonious  conception  of  all  the  various  observa- 
tions, but  also  to  furnish  a  scientific  basis  for  a  further  successful 
development  of  the  subject. 

1  Berlin,  klin.  Wochenschr.  No.  44,  1905,  and  No.  3,  1906. 


XLIL    THE  MULTIPLICITY  OF  ANTIBODIES  OCCURRING 
IN  NORMAL  SERUM.1 

By  Dr.  MAX  NEISSER,  Member  of  the  Institute. 

FOLLOWING  the  fundamental  researches  made  by  Fliigge  and 
Buchner  and  their  pupils  on  the  bactericidal  power  of  normal 
blood,  we  have  come  to '  recognize  a  large  number  of  properties 
possessed  by  normal  serum.  According  to  our  present  knowledge 
we  must  regard  these  properties  as  due  to  the  presence  of  anti- 
bodies in  the  broadest  sense. 

Thus  far  the  only  theory  which  has  satisfactorily  accounted 
for  the  origin  of  these  antibodies,  from  a  physiological  standpoint 
and  without  invoking  the  aid  of  teleological  "protective  substances," 
is  Ehrlich's  Side-chain  Theory.  According  to  this  the  cells  of  the 
organism  produce  substances,  side-chains,  whose  physiological 
function,  so  long  as  they  are  part  of  the  cell,  is  to  lay  hold  of 
certain  foodstuffs.  Side-chains  thus  anchored  are  replaced  by  the 
cell,  and  when  this  regeneration  is  excessive,  the  surplus  side 
chains  are  thrust  off  into  the  blood.  As  a  result  of  this,  the  blood 
serum  contains  a  large  number  of  different  side-chains.  For 
example,  one  variety  of  these  side-chains  may  happen  to  have  an 
affinity  for  a  particular  toxin;  it  will  be  found  possible,  by  care- 
fully injecting  this  toxin,  to  increase  the  regeneration  and  thrusting- 
off  to  an  extraordinary  degree,  and  thus  an  immunity  is  produced 
against  that  toxin.  From  this  standpoint,  then,  immunity  is 
regarded  as  merely  a  quantitative  increase  in  the  exercise  of  a 
normal  function. 

This  view  has  important  bearings  on  our  conception  of  the 
antibodies  occurring  in  normal  serum.  It  is  apparent  that  the 
diversity  of  the  antibodies  which  can  be  produced  artificially,  is 
entirely  analogous  to  the  variety  of  antibodies  present  normally. 

1  Reprinted  from  Deutsche  med.  Wochenschr.,  No.  49,  1900. 

587 


588  COLLECTED  STUDIES   IN   IMMUNITY. 

This  plurality  of  normal  antibodies,  advocated  by  Ehrlich  in 
a  number  of  papers  (8,  9,  ii);  is  strongly  combated  by  Bordet  by 
Buchner,  who  adhere  to  a  Unitarian  conception.  These  authors  (5) 
agree  that  hsemolysins  and  bacteriolysins  are  made  up  of  two 
parts;  while  they  admit  that  the  "interbody"  is  different,  they 
insist  that  only  a  single  ferment-like  substance,  the  "alexin, "  is 
involved  in  the  lysis  of  all  the  various  species  of  blood  or  bacterial 
cells. 

Kraus  (12)  goes  still  further.  He  found  that  rabbit  erythrocytes 
could  be  protected  by  normal  horse  serum  against  several  different 
blood  poisons,  and  concluded  "that  any  given  hsemolytic  poison 
acting  on  rabbit  blood,  can  be  paralyzed  in  its  action  by  means 
of  normal  horse  serum." 

In  view  of  the  theoretical  importance  of  this  subject,  we  have 
thought  it  advisable  to  study  the  question  of  the  unity  or  plurality 
of  normal  antibodies.  In  doing  this  we  have  studied  experiments 
already  reported  and  have  supplemented  these  with  some  observa- 
tions of  our  own. 

So  far  as  the  hsemolysins  are  concerned,  it  has  long  been  known 
that  many  sera  have  the  power  to  dissolve  the  blood-cells  of  a 
number  of  other  species.  It  is  only  recently,  however,  that  we 
have  learned  how  easy  it  is  to  produce  artificial  hsemolysins  by 
immunization.  The  specificity  of  these  artificial  hsemolysins  was 
first  demonstrated  by  Bordet  (2),  but  it  was  not  until  Ehrlich 
and  Morgenroth  devised  elective  absorption  tests  do)  that  the 
subject  became  clear.  This  procedure  is  based  on  Ehrlich's  con- 
ception of  a  chemical  union  of  erythrocytes  and  hsemolysin;  it 
consists  in  saturating  a  serum  which  contains  several  hsemolysins, 
with  erythrocytes  of  one  of  the  species,  under  conditions  which 
prevent  the  solution  of  these  cells.  Under  these  circumstances 
the  erythrocytes  combine  with  their  specific  hsemolysin,  and 
abstract  it  'from  the  fluid.  On  centrifugirig,  it  is  found  that  the 
fluid  contains  only  the  remaining  hsemolysins,  and  these  have  not 
diminished  in  amount.  By  means  of  this  procedure,  Ehrlich  and 
Morgenroth  UD  demonstrated  the  existence  of  several  distinct 
specific  hsemolysins  in  a  normal  serum.  They  showed  that  a  normal 
goat  serum  which  dissolved  the  blood-cells  of  guinea-pigs  and 
rabbits,  could  be  freed  from  one  of  these  hsemolysins  by  treatment 
with  the  corresponding  blood-cells,  the  other  hsemolysins  remaining 
unaffected.  It  is  to  be  noted,  however,  that  the  hsemolysins  consist 


MULTIPLICITY   OF  ANTIBODIES  IN   NORMAL  SERUM.      589 

of  two  parts,  which  Ehrlich  terms  interbody  and  complement 
respectively.  The  interbody  combines  with  the  erythrocyte  on  the 
one  hand,  and  with  the  active  dissolving  agent,  the  complement, 
on  the  other.  The  experiments  just  described,  therefore,  demon- 
strated merely  the  plurality  of  the  interbodies,  and  shed  no  light 
on  the  unity  or  plurality  of  the  complements.  In  fact  it  was 
easily  conceivable  that  a  single  complement  (the  alexin  of  Buchner 
and  Bordet)  fitted  to  both  interbodies  and  effected  the  solution 
of  both  species  of  erythrocytes.  Ehrlich  and  Morgenroth,  however, 
were  able  to  demonstrate  that  the  complements  concerned  were 
different.  They  filtered  a  serum  through  Pukall  filters,  and  so 
effected  a  separation  of  the  two,  one  of  the  complements  passing 
through  completely,  while  all  but  traces  of  the  other  were  held 
back.  It  was  thus  shown  that  the  hsemolytic  "  power  "  of  the  normal 
goat  serum  against  rabbit  and  guinea-pig  blood  was  due  to  at 
least  four  distinct  substances  existing  independently  in  the  serum 
side  by  side. 

Xuttall  (17>  was  able  to  show  that  normal  rabbit  blood  was 
bactericidal  for  B.  anthrax,  B.  subtilis,  and  Bact.  megatherium; 
Nissen  (16)  demonstrated  the  bactericidal  power  of  rabbit  blood 
on  cholera  and  typhoid  bacilli,  and  on  coccus  aquatilis,  and 
Buchner  (4)  found  that  cell-free  blood  serum  of  rabbits  acted 
on  anthrax,  erysipelas  of  swine,  typhoid  bacilli,  cholera,  etc. 
There  is  considerable  variation  in  the  action  of  the  serum,  on 
different  bacteria.  Thus  Nuttall  found  that  rabbit  blood  acted 
on  anthrax  bacilli,  but  not  on  staphylococcus  aureus.  On  the 
other  hand,  different  sera  behave  differently  on  the  same  species 
of  bacterium.  Thus  Buchner  found  ox  and  horse  serum  without 
effect  on  typhoid  bacilli.  The  question  again  arises,  whether  the 
bactericidal  action  of  normal  sera  is  due  to  a  single  substance  or 
to  different  substances. 

Experiments  to  decide  this  question  were  made  by  Nissen  <16), 
although  it  must  be  admitted  that  they  were  not  entirely  conclusive. 
He  injected  a  rabbit  intravenously  with  large  quantities  of  the 
coccus  aquatalis  and  observed  that  the  blood  obtained  immediately 
after  had  lost  its  bactericidal  power  for  this  coccus,  while  the 
bactericidal  power  for  cholera  and  typhoid  bacilli  remained 
unchanged. 

Extensive  investigations  concerning  this  point  were  then  made 
by  Bail  d)  who  employed  the  absorption  test.  He  found  on 


590  COLLECTED  STUDIES  IN  IMMUNITY. 

on  adding  dead  staphylococci  in  not  too  large  quantity  to  rabbit 
serum,  that  the  clear  fluid  separated  by  the  centrifuge  was  still 
bactericidal  for  typhoid  bacilli,  but  not  for  staphylococci.  The 
test  also  succeeded  when  done  vice  versa,  and  with  staphylococci 
and  cholera,  as  well  as  with  typhoid  and  cholera  bacilli. 

By  .means  of  the  absorption  test  I  was  able  to  demonstrate 
that  the  bactericidal  substances  of  normal  rabbit  serum  were 
independent  of  the  hsemolytic  substances.  Thus,  on  adding  anthrax 
bacilli  to  normal  rabbit  serum,  and  then  centrifuging,  it  was  possible 
to  remove  the  bactericidal  power  against  anthrax  without  in  any 
way  impairing  the  hsmolytic  power  of  the  serum  for  goat  and 
sheep  blood-cells. 

From  what  has  been  said  it  will  bs  seen  that  the  bactericidal 
action  which  normal  rabbit  serum  exerts  on  different  species  of 
bacteria  is  found,  by  experiment,  to  be  due  to  several  distinct 
substances  in  no  way  dependent  on  one  another. 

In  the  case  of  another  class  of  antibodies,  the  agglutinins,  recent 
investigations  have  shown  that  they  too  may  exist  preformed  in 
normal  serum.  Here  again  the  question  arose  whether  but  a 
single  substance  was  concerned,  or  whether  there  were  many 
different  substances. 

The  first  experiments  in  this  direction  were  made  by  Bordet  (3), 
who  studied  normal  horse  serum.  This  has  the  power  to  agglu- 
tinate cholera  and  typhoid  bacilli.  By  means  of  the  absorption 
technique  of  Ehrlich  and  Morgenroth,  Bordet  found  that  after 
centrifuging  serum  which  had  been  saturated  with  one  of  the 
organisms,  the  agglutinating  power  for  that  organism  would  have 
been  lost,  while  that  for  the  other  organism  would  still  be  present, 
and  vice  versa.  Subsequently  Malkoff  (14)  reported  similar  results 
with  red  blood-cells.  He  found  that  normal  goat  serum  agglu- 
tinated (without  dissolving)  the  erythrocytes  of  the  rabbit, 
pigeon,  and  man,  while  the  erythrocytes  of  other  animals  were 
but  little  or  not  at  all  agglutinated.  Furthermore,  it  was  found 
that  there  was  considerable  individual  fluctuation  in  the  serum 
of  different  goats.  Working  with  the  goat  serum,  which  agglu- 
tinated the  three  bloods  just  mentioned,  he  found  that  by  adding, 
for  example,  pigeon  erythrocytes  and  then  centrifuging,  the  cen- 
trifuged  serum  would  have  lost  its  agglutinating  power  for  pigeon 
erythrocytes,  but  was  still  able  to  agglutinate  the  other  two  species 
of  blood- cells.  The  experiment  succeeded  in  all  possible  com- 


MULTIPLICITY   OF  ANTIBODIES   IN  NORMAL  SERUM.      591 

binations,  so  that  even  when  two  species  of  blood-cells  were 
added  at  once,  the  agglutinating  power  for  these  could  be  reduced 
to  nil  while  the  power  for  the  remaining  species  of  blood  was  unim- 
paired. We  see,  therefore,  that  the  results  are  entirely  similar  to 
those  obtained  with  the  haBmolysins  and  bacteriolysins;  the 
agglutinating  power  of  normal  serum  on  different  species  of  cells  is 
due  to  separate  and  distinct  substances  contained  in  the  serum. 

In  addition  to  the  foregoing  we  may  also  consider  for  a  moment 
those  antibodies  which  act,  not  on  bacteria  or  blood-cells,  but 
on  ferments  and  toxins,  in  other  words,  the  antitoxins  and  anti- 
ferments.  These  bodies  are  not  known  directly,  but  only  indi- 
rectly by  their  neutralizing  effect;  we  know  little  about  their  occur- 
rence in  normal  serum.  Landsteiner  d3);  citing  also  the  older 
literature,  found  antitryptic  substances  in  normal  rabbit,  guinea- 
pig,  and  ox  serum.  Morgenroth  (is)  found  antibodies  against 
rennin  and  against  cynarase  in  the  serum  of  normal  goats  and 
horses.  By  specific  immunization  this  investigator  was  able  to 
show  that  rennin  and  cynarase  were  two  distinct  ferments,  and 
that  the  antirennin  of  normal  serum  was  distinct  from  the  normal 
anticynarase.  Morgenroth  found  that  the  relative  amounts  of  the 
two  antibodies  differed  in  two  horse  sera  which  he  investigated. 

The  existence  of  normal  antitoxins  has  also  been  reported. 
Meade  Bolton,  and  later  Cobbett  (6)  found  that  a  considerable 
proportion  of  normal  horses  had  diphtheria  antitoxin  in  their 
serum,  and  that  the  amount  of  this  was  very  variable.  Wasser- 
mann  (i»)  found  that  not  a  few  normal  human  individuals  had 
diphtheria  antitoxin  in  their  blood.  Ehrlich  (7)  encountered  a 
normal  horse  serum  which  contained  an  antibody  against  teta- 
nolysin,  and  Krauss  (12)  found  normal  horse  serum  effective  against 
a  number  of  hsemolysins.  In  a  paper  which  Dr.  Wechsberg  and 
I  hope  soon  to  publish,  it  will  be  shown  that  we  have  constantly 
found,  in  normal  human  serum,  an  antibody  against  staphylotoxin. 

In  view  of  the  fact  that  horse  serum  protects  rabbit  erythro- 
cytes  against  tetanolysin,  staphylolysin,  and  other  haemolysins, 
Krauss  concludes  that  the  protective  action  is  due  to  a  single 
substance  in  horse  serum,  and  then  concludes  further  that  these 
haBmolysins  differ  only  quantitatively  and  not  qualitatively.  A  few 
exact  quantitative  experiments  would  have  convinced  Krauss  that 
this  assumption  of  the  non-specificity  of  haemolysins  is  absolutely 
incorrect.  It  can  be  shown  that  an  antistaphylolysin,  artificially 


592 


COLLECTED   STUDIES   IN  IMMUNITY. 


produced  by  immunizing  rabbits,  protects  only  against  staphylolysin, 
and  not  against  tetanolysin.  This  is  well  shown  in  the  paper 
about  to  be  published  by  us.  So  also  it  can  be  shown  that  a  tetanus 
antitoxin  derived  from  a  horse  has  a  marked  protective  action 
against  tetanolysin,  whereas  the  protective  action  against  staphy- 
lolysin is  no  greater  than  that  of  normal  horse  serum.  Finally,  it 
can  be  shown  that  normal  horse  serum  usually  protects  against 
tetanolysin  and  staphylolysin,  but  not  against  the  hsemolysin  of 
normal  goat  serum.  The  last-named,  it  will  be  remembered,  acts 
on  rabbit  blood-cells.  These  haemolytic  poisons,  therefore,  differ 
qualitatively  from  one  another. 

We  see,  then,  that  the  antibody  present  in  normal  horse  serum 
does  not  protect  rabbit  erythrocytes  against  all  blood  poisons,  for 
it  is  not  able  to  prevent  the  solvent  action  of  normal  goat  serum. 
Furthermore,  it  will  be  seen  from  the  following  experiment  that 
the  protective  action  against  a  number  of  different  blood  poisons 
is  not  due  to  a  single  substance.  The  blood  poisons  employed 
were  tetanolysin  and  staphylolysin,  and  the  serum  of  four  normal 
horses  was  tested  against  these  quantitatively.  To  begin,  it  was 
necessary  to  determine  the  complete  solvent  dose  of  tetanolysin 
and  of  staphylolysin  for  one  drop  of  rabbit  blood.  Then  the 
amount  of  horse  serum  which  sufficed  to  completely  neutralize 
(inhibit)  this  dose  was  determined.  The  following  is  an  abbre- 
viated protocol  of  such  an  experiment. 

The  complete  solvent  dose  of  the  staphylolysin  employed  (14-day 
filtered  bouillon  culture  of  staphylococcus  pyogenes  aureus)  was 
0.05  cc.  for  one  drop  of  rabbit  blood.  The  solvent  dose  of  tetan- 
olysin was  0.25  cc. 


TABLE  I. 


No.  of  cc.  which  entirely  Neutral- 
izes the  Effect  of  a  Complete 
Solvent  Dose  of  Staphylolysin. 

No.  of  cc.  which  Entirely  Neu- 
tralize the  Complete 
Solvent  Dose  of  Tetanolysin. 

Horse  serum  1  
Horse  serum  2        .... 

0.025 
0  075 

0.25 
0  05 

Horse  serum  3  

0  025 

more  than  1 

Horse  serum  4  

0.25 

0.25 

That  is  to  say,  the  number  of  doses  of  antibody  contained  in 
each  cubic  centimeter  was 


MULTIPLICITY  OF  ANTIBODIES  IN  NORMAL  SERUM.      593 


For  horse  serum  1 

' '    horse  serum  2 

'    horse  serum  3 

' '    horse  serum  4  .  . 


Antistaphylolysin. 
40 
13.3 
40 
-4 


Antitetanolysin. 

'4 

20 

less  than  1 


Compared  to  each  dose  of  antitetanolysin  there  were  in 


Horse  serum  1 . 
Horse  serum  2. 
Horse  serum  3. 
Horse  serum  4. 


10  doses  antistaphylolysin 
0.67" 
more  than  40  doses  antistaphylolysin 


Such  a  result,  however,  can  be  explained  only  by  assuming  the 
existence  of  two  different  antibodies. 

The  point  is  proved  by  another  experiment.  To  a  given  speci- 
men of  horse  serum  whose  antitoxic  power  for  staphylolysin  is 
known,  enough  staphylolysin  is  added  to  completely  satisfy  the 
antistaphylolysin.  When  this  has  been  done  it  will  be  found 
that  the  antitoxic  power  for  tetanolysin  has  not  been  affected. 

Thus  we  see  that  wherever  the  bactericidal,  haemolytic, 
agglutinating,  antif ermantative,  and  antitoxic  "  powers "  of  normal 
sera  are  carefully  analyzed,  they  are  found  to  be  due  to  separate 
independent  substances  for  each  action.  By  this  we  do  not  mean 
to  say  that  the  origin  of  these  substances  is  necessarily  to  be 
ascribed  to  the  action  of  the  elements  against  which  they  are 
found  to  be  directed.  On  the  contrary,  for  many  of  these  sub- 
stances, e.g.,  diphtheria  antitoxin  in  normal  horses,  it  seems  likely 
that  certain  normal  "side-chains"  of  whose  physiological  purpose 
we  are  still  entirely  ignorant  happen  to  have  affinity  to  a  group 
possessed  by  some  bacterium,  ferment,  or  toxin. 

The  presence  of  an  antibody  in  normal  serum  merely  proves 
that  the  animal  somewhere  possesses  certain  chemical  groups, 
receptors,  which  happen  to  have  an  affinity  to  the  bacterium  in 
question;  and  that  normally  there  is  a  moderate  overproduction 
of  these  receptors  with  a  consequent  appearance  of  thrust-off 
receptors  in  the  blood. 

This  thrusting-off,  then,  is  a  physiological  process  which  we  are 
able  to  influence  by  immunization.  As  a  result  of  this  there  is  a 
sudden  enormous  overproduction  of  one  particular  receptor,  a  kind 
of  pure  culture  of  the  receptor  grown  in  the  animal.  It  is  obvious, 
however,  that  wherever  we  are  able  by  immunization  to  cause  an 
excessive  thrusting-off  of  a  receptor,  there  also  will  it  be  possible 


594  COLLECTED   STUDIES   IN  IMMUNITY. 

for  such  receptors  to  be  thrust  off  normally.  In  view  of  the  great 
diversity  of  substances  which  we  are  able  to  produce  by  artificial 
immunization,  it  should  not  surprise  us  to  encounter  a  great  variety 
of  substances  in  normal  serum.  When  we  consider,  further,  how 
varied  is  the  behavior  of  different  species  and  even  of  different 
individuals  of  the  same  species,  we  shall  at  once  associate  this  with 
the  great  divergence  in  the  content  of  normal  antibodies  in  different 
species  and  different  individuals.  As  a  matter  of  fact  these  varia- 
tions are  no  greater  than  the  variations  in  hairiness  or  in  pigmen- 
tation. 

Further  experimental  investigations  will  surely  reveal  the  presence 
of  many  more  antibodies  in  normal  serum,  and  it  is  possible  that 
additional  clinico-experimental  studies  may  even  give  us  the 
,key  to  their  physiological  function.  An  insight  into  their  signifi- 
cance in  man  might  open  up  new  ways  in  diagonosis  and  therapy. 

LITERATURE. 

1.  BAIL,  Archiv  fur  Hygiene,  1899,  Vol.  XXV,  page  284. 

2.  BORDET,  Annales  Pasteur,  Vol.  XII,  No.  10. 

3.  BORDET,  Annales  Pasteur,  1899,  Vol.  XIII,  page  225. 

4.  BUCHNER,  Centralblatt  Bacteriologie,  Vol.  V,  page  817;    Vol.  VI,  page  1. 

5.  BUCHNER,  Miinchener  med.  Wochenschr.  1900,  Nos.  9  and  35. 

6.  COBBETT,  Centralblatt  Bacteriologie,  1899,  Vol.  XXVI,  page  548. 

7.  EHRLICH,  Berlin,   klin.    Wochenschr.  1898,  No.  12  (Ges.  d.  Charite"  Aerzte, 

Feb.  3.) 

8.  EHRLICH,  *The  Croonian  Lecture,  Proceed.  Royal  Soc.,  Vol.  LXVI,  page  424. 

9.  EHRLICH,  Semaine  medicale,  Dec.  6,  1899. 

10.  EHRLICH  and  MORGENROTH,  Berlin,  klin.  Woshenshcr.  1900,  No.  1. 

11.  EHRLICH  and  MORGENROTH,  Berlin,  klin.  Wochenschr.  1900,  No.  31. 

12.  KRAUSS,  Wiener  klinische  Wochenschr.  1900,  No.  3. 

13.  LANDSTEINER,    Centralblatt    Bacteriologie,    1899,    Vol.    XXV,    page    546; 

Vol.  XXVII,  page  357. 

14.  MALKOFF,  Deutsche  med.  Wochenschr.  1900,  page  229. 

15.  MORGENROTH,    Centralblatt    Bacteriologie,    Vol.    XXVI,    page    349;     Vol. 

XXVII,  page  721. 

16.  NISSEN,  Zeitschrift  Hygiene,  Vol.  VI,  page  487. 

17'.  NUTTALL,  Zeitschrift  Hygiene,  1888,  Vol.  IV,  page  353. 
18.  WASSERMANN,  Zeitschrift  Hygiene,  Vol.  XIX,  page  408. 


XLIII.    THE  BINDING  OF  ILEMOLYTIC  AMBOCEPTORS.i 

By  Dr.  J.  MORGENROTH,  Member  of  the  Institute. 

IT  has  been  established  that  the  haemolytic  amboceptors  are 
bound  by  the  blood-cell  receptors  for  which  they  have  a  specific 
affinity.  In  earlier  papers2  it  was  experimentally  shown  that  the 
amount  of  amboceptor  which  can  be  bound  by  the  blood-cells 
varies  to  an  extraordinary  degree.  We  call  an  "amboceptor 
unit," 3  the  amount  of  amboceptor  which  suffices  to  dissolve  a 
certain  quantity  of  red  blood-cells  (1  cc.  5%  suspension)  when 
plentiful  amount  of  complement  is  present.  Experience  has  shown 
that  the  combining  capacity  of  the  blood-cells  varies  from  one  to 
one  hundred  amboceptor  units.  On  centrifuging  the  blood-cells 
after  these  have  bound  the  amboceptor,  and  resuspending  them  in 
salt  solution,  it  will  be  found  that  the  amboceptor  remains  bound 
unchanged,  and  is  not  given  off  to  the  fluid  in  demonstrable  quan- 
tities at  room  temperature.  It  was  natural  to  investigate  the 
firmness  of  this  amboceptor  union  in  suitable  cases,  namely,  cases 
in  which  a  multiple  of  the  amboceptor  unit  had  been  anchored. 

By  repeated  centrifuging  and  resuspension  hi  salt  solution,  it 
is  possible  to  obtain  blood-cells  laden  with  amboceptor  in  a  medium 
entirely  free  from  recognizable  traces  of  amboceptor.  A  curious 
phenomenon  is  observed  when  fresh  blood-cells  of  the  same  species 
are  added  to  such  a  suspension.  After  a  time  some  of  the  ambo- 
ceptors originally  bound  to  the  blood-cells  pass  over  to  the  new 

1  Reprinted,  from   Munchener  med.  Wochenschrift,  No.  2,   1903.     A  more 
recent  discussion  of  this  subject  by  the  same  author  will  be  found   in  Biochem.  • 
Zeitschrift.  Vol.  XX,  1909. 

2  Ehrlich  and  Morgenroth,  Berliner  klin.  Wochenschr.  No.  10,  1901.     This 
volume,  page  71.     See  also  Ehrlich,  in  Nothnagel's  Spez.  Pathologic  u.  Therapie, 
Vol.  VIII. 

'Morgenroth  and  Sachs,  Berliner  klin.  Wochenschrift,  No.  35,  1902.  This 
volume,  p.  254. 

595 


596  COLLECTED  STUDIES   IN  IMMUNITY. 

blood-cells,  so  that  finally  all  the  blood-cells  in  the'  mixture  contain 
an  amount  of  amboceptor,  sufficient,  when  suitable  amounts  of 
complement  are  added,  to  produce  complete  solution  of  the  entire 
mixture.  This  is  shown  by  the  following  experiments. 

To  20  cc.  5%  serum-free  suspension  of  ox  blood-cells  one  adds 
4.0  cc.  inactive  serum  of  a  rabbit  immunized  against  ox  blood. 
The  complete  solvent  dose  of  this  immune  serum  (for  1  cc.  5%  sus- 
pension) when  0.1  cc.  guinea-pig  serum  is  added  as  complement, 
is  0.0015  cc.  The  amount  employed  in  this  experiment  therefore 
contained  130  amboceptor  units.  The  mixtures  were  kept  at  38° 
for  one  hour,  and  frequently  shaken.  The  blood-cells  were  sepa- 
rated by  centrifuge,  and  washed  three  times  with  40  cc.  salt  solution, 
and  then  made  up  to  the  volume  of  the  original  suspension.  The 
last  wash  water  was  free  from  amboceptor.  One  cc.  of  this  sus- 
pension was  mixed  with  one  cc.  of  a  fresh  5%  suspension  of  ox 
blood-cells,  and  the  mixtures  kept  for  one  hour  in  a  water-bath 
at  40°.  On  adding  0.2  cc.  guinea-pig  serum,  it  was  found  at  the 
end  of  fifteen  minutes  that  complete  solution  of  the  entire  quantity 
of  blood  had  ensued. 

This  shows  that  in  the  course  of  one  hour  at  40°,  the  blood-cells 
added  afterwards  had  absorbed  at  least  sufficient  amboceptor  to 
effect  solution.  Similar  experiments  with  blood-cells  laden  with 
3,  6,  10,  and  60  times  the  amboceptor  unit  yielded  entirely  analogous 
results.  The  action  takes  place  even  at  0°  C.,  though  much  more 
slowly. 

The  result  of  these  experiments  is  apparently  at  variance  with 
earlier  statements,  that  the  fluid  is  free  from  amboceptors.  It  is 
obvious  that  the  amboceptors  can  only  get  from  one  blood-cell 
to  another  by  way  of  the  fluid  medium.  The  contradiction,  how- 
ever, is  explained  by  assuming  that  the  fluid  is  not  absolutely  free 
from  amboceptors,  but  contains  such  minute  traces  that  they 
escape  detection.  When,  in  the  experiment,  the  blood-cells  subse- 
quently added  combine  with  the  amboceptors  present  in  the  fluid, 
conditions  are  produced  whereby,  in  accordance  with  the  law  of 
chemical  equilibrium,  additional  small  traces  of  amboceptor  are 
liberated  into  the  fluid.  With  the  anchoring  of  this  by  the  fresh 
blood-cells,  the  process  is  repeated,  so  that  the  latter  bind  more 
and  more  amboceptor. 

In  the  binding  of  the  amboceptors  we  are  therefore  dealing 
with  a  reversible  process  in  which  the  equilibrium  is  such  that 


BINDING  OF   H.EMOLYTIC  AMBOCEPTORS.  597 

the  quantity  of  amboceptor  in  solution  is  usually  too  minute  to 
be  detected.  Similar  conditions  in  the  solution  have  recently  been 
described  for  the  hsemolytic  substances  of  certain  organ  extracts. 
These  substances  are  only  very  slightly  soluble  hi  salt  solution. 
Nevertheless,  when  susceptible  blood-cells  are  present  at  the  same 
time,  the  substances  are  anchored  by  the  cells,  i.e.,  abstracted 
from  the  solution,  while  a  further  minute  quantity  is  given  off  to 
the  solution.  In  connection  with  the  experiments  made  at  that 
time,1  we  called  attention  to  the  analogy  existing  between  this 
phenomenon  and  certain  occurrences  in  dyeing. 

It  was  necessary,  now,  to  determine  how  the  complete  ha3moly- 
sin,  i.e.,  amboceptor  plus  complement,  would  behave  in  an 
experiment  of  this  kind.  The  result  was  highly  interesting,  for 
it  was  found  that  the  ability  of  the  amboceptor  to  pass  from  the 
receptor  of  one  blood  corpuscle  to  that  of  another  existed  only 
so  long  as  the  amboceptor  had  not  also  combined  with  comple- 
ment. On  adding  immediately  a  suitable  amount  of  complement 
to  mixtures  of  blood-cells  laden  with  amboceptor  and  fresh  blood- 
cells,  it  will  be  found  that  only  the  former  are  dissolved,  i.e.,  only 
half  of  the  mixture.  Even  when  the  complement  is  added  after 
10,  20,  or  40  minutes,  only  part  of  the  blood-cells  is  dissolved.  It 
is  only  when  the  complement  is  not  added  until  after  sixty  minutes 
have  elapsed,  i.e.,  after  time  has  been  given  to  permit  the  passage 
of  sufficient  amboceptor,  that  complete  haemolysis  occurs. 

Twenty  cc.  of  a  5%  suspension  of  ox  blood-cells  freed  from 
serum  are  mixed  with  0.048  cc.  of  the  inactive  immune  serum  =16 
amboceptor  units.  The  mixture  is  kept  at  38°  and  frequently  shaken, 
after  which  the  blood-cells  are  separated  by  centrifuging.  The 
blood-cells  are  washed  three  times  with  salt  solution  until  the 
wash  water  is  entirely  free  from  amboceptor.  After  making  the 
suspension  up  to  the  original  volume,  1  cc.  doses  are  mixed  with 
1  cc.  doses  of  a  fresh  5%  suspension  of  ox  blood-cells.  The  mixtures, 
kept  in  a  water-bath  at  40°  each,  received  0.2  cc.  doses  of  guinea-pig 
serum  at  different  intervals,  namely,  at  once,  and  after  10,  20, 
40,  and  60  minutes.  In  order  to  produce  the  maximum  ha3molytic 
effect,  all  the  tubes  were  kept  in  the  water-bath  for  three  hours. 
At  the  end  of  that  time,  half  of  the  blood-cells,  corresponding  to 


1  Korschun  and  Morgenroth,  Berliner  klin.  Wochenschrift,  No.  37,   1902. 
This  volume,  page  267. 


598 


COLLECTED  STUDIES   IN   IMMUNITY. 


the  1  cc.  of  sensitized  blood-cells,  had,  of  course,  dissolved.  The 
degree  of  solution  which  the  other  half  had  undergone,  varied 
with  the  length  of  time  after  which  the  complement  was  added, 
and  is  shown  in  the  accompanying  table: 


Complement  Added. 

Degree  of  Solution. 

1 

2 
3 
4 
5 

at  once 
after  10  minutes 

"     20       " 
"     30       " 
"     60       " 

0  to  slight 
slight  to  moderate 
moderate 
strong 
complete 

On  subsequently  adding  a  further  0.2  cc.  guinea-pig  serum  to 
tubes  1-4,  and  placing  them  in  the  water-bath,  complete  solution 
was  produced. 

It  is  not  difficult  to  explain  this  phenomenon.  On  adding 
complement  to  mixtures  of  sensitized  and  fresh  blood-cells,  the 
complement  is  rapidly  bound  by  the  anchored  amboceptors.  We 
know  from  earlier  experiments  that  these  have  an  increased  affinity 
for  the  complement.1  If  the  amount  of  complement  is  relatively 
small,  while  that  of  the  anchored  amboceptors  is  large,  it  is  obvious 
that  only  part  of  the  amboceptors  will  be  occupied  by  complement. 
The  anchored  amboceptors  which  have  bound  complement  are 
evidently  no  longer  able  to  let  go  of  their  receptor.  This  fact 
shows  that  the  anchoring  of  the  complementophile  group  of  the 
amboceptor  produces  an  increase  in  the  binding  power  of  the 
cytophile  group.  The  anchored  amboceptors  which  are  uncom- 
bined  with  complement,  naturally  retain  their  freedom  of  move- 
ment, and  are  thus  enabled  to  pass  over  to  the  freshly  added  blood- 
cells.  This  is  demonstrated  by  the  occurrence  of  haemolysis  on 
the  further  addition  of  complement. 

We  believe  that  these  experiments  constitute  an  important 
addition  to  our  knowledge  of  the  relations  existing  among  ambo- 
ceptor, receptor,  and  complement.  From  a  well-known  experiment 
made  by  Bordet,2  we  know  that  after  haemolysis  has  begun,  ambo- 
ceptor and  complement  remain  permanently  combined.  Bordet 

1  Ehrlich  and  Morgenroth,  Berliner  klin.  Wochenschr.,  No.  1,   1899.     This 
volume,  page  1. 

2  Bordet,  Annales  Pasteur,  No.  5,  1901. 


BINDING  OF   H^MOLYTIC  AMBOCEPTORS.  599 

determined  the  quantity  of  blood-cells  which  would  just  be 
completely  dissolved  by  a  hsemolytic  serum  when  the  cells  were 
added  at  once.  He  then  divided  the  blood-cells  into  two  equal 
parts,  added  one  part  and  then  the  other  after  the  first  had  been 
haemolyzed.  The  second  portion  remained  undissolved.  Bordet 
incorrectly  interpreted  this  as  indicating  a  physical  adsorption 
of  the  amboceptor,  but,  as  already  indicated,1  the  phenomenon  is 
due  to  the  fact  that  the  blood-cells  bind  multiples  of  the  amboceptor 
unit. 

Attempts  to  liberate  bacterial  agglutinins  from  their  combina- 
tion with  the  cells  were  made  some  time  ago  by  Hahn  and  Tromms- 
dorff.2  These  investigators  treated  agglutinated  bacteria  with 
weakly  alkaline  and  weakly  acid  solutions  and  actually  succeeded 
in  liberating  a  portion  of  the  bound  agglutinin.  The  agglutinin 
so  liberated  was  still  active.  More  recently  Landsteiner3  succeeded 
in  liberating  the  agglutinin  from  agglutinated  blood  corpuscles  by 
digestion  with  physiological  salt  solution  at  50°.  This  author, 
moreover,  found  that  even  at  lower  temperatures  a  certain  amount 
of  agglutinin  passed  into  the  salt  solution  used  for  washing  the  agglu- 
tinated cells,  and  he  therefore  concludes,  probably  correctly,  that 
the  combination  of  cell  and  agglutinating  substance  decomposes 
even  at  ordinary  temperatures,  though  to  a  less  degree  than  at 
higher  temperatures. 

It  is  necessary  constantly  to  call  attention  to  the  significance 
of  the  chemical  union  of  the  amboceptors  for  a  correct  understanding 
of  the  fundamental  principles  of  the  immunity  reactions.  We 
are  here  dealing  with  a  chemical  binding  which  is  unaccompanied 
by  any  toxic  action 'whatever,  but  which  at  any  time,  through  the 
addition  of  complement,  can  become  manifest  by  such  action.  Just 
this  makes  it  possible  to  demonstrate  the  essential  distinction 
between  the  chemical  binding  and  toxic  action,  a  distinction  which 
finds  its  expression  in  the  separation  of  the  toxin  molecule  into  a 
toxophore  and  a  haptophore  group.  Gruber  and  Durham4  were 
the  first  to  demonstrate  the  fact  that  cholera  vibrios  could  remove 
cholera-immune  bodies.  Since,  however,  they  identified  these 

1  Ehrlich  and  Morgenroth,  loc.  cit. 

2  Hahn  and  Trommsdorff,  Miinchener  med.  Wochenschrift,  No.  13,  1900. 

3  Landsteiner,  Wiener  klin.  Rundschau,  No.  40,  1902,  and    Munch,    med. 
Wochenschrift,  No-.  46,  1902. 

4  Gruber,  Wiener  klin.  Wochenschrift,  No.  12,  1896. 


600  COLLECTED  STUDIES  IN  IMMUNITY. 

bodies  with  the  agglutinins,  they  could  merely  conclude  that  the 
agglutinins  were  used  up  in  the  reaction.  That  a  substance  is  used 
up  as  a  result  of  its  action,  is  however,  self  evident,  and  constitutes 
the  basis  of  all  dosage.  If  this  were  not  so  we  should  be  able  with 
any  poison  to  produce  an  endless  toxic  action,  just  as  theoretically 
ferment  action  can  go  on  indefinitely.  Although  of  great  impor- 
tance in  itself,  all  that  Gruber  demonstrated  was  the  fact  that  treat- 
ment with  specifically  acting  agencies  caused  the  substances  to 
disappear.  An  insight  into  the  nature  of  this  process,  particularly 
whether  it  was  a  destruction  or  merely  a  binding,  would  have  required 
a  further  systematic  analysis,  and  this  was  not  undertaken.  More- 
over, just  this  analysis  would  have  been  extremely  difficult,  because 
of  the  views  then  and  perhaps  still  held  by  Gruber  1>  namely,  that 
agglutinins  and  bacteriolysins  are  identical. 

1  Gruber,  Munchener  med.  Wochenschrift,  No.  48,  1901. 


XLIV.    THE  JOINT  ACTION  OF  NORMAL  AND  IMMUNE 
AMBOCEPTORS  IN  HAEMOLYSIS,1 

By  Dr.  HANS  SACHS. 

PFEIFFER  AND  FRIEDBERGER2  have  recently  published  some 
very  interesting  observations  concerning  the  antibacteriolytic 
action  of  normal  sera.  They  find,  for  example,  that  normal  sera 
which  in  themselves  possess  no  antilytic  power,  acquire  such  power 
on  digesting  them  with  bacteria.  Curiously  also,  the  sera  thus 
treated  become  specifically  antilytic,  so  that  a  serum  treated  with 
cholera  vibrios  acquires  inhibiting  properties  only  against  the  bacte- 
riolysis of  these  organisms;  a  serum  treated  with  typhoid  bacilli 
protects  only  typhoid  bacilli  against  bacteriolysis. 

How  is  this  action  to  be  explained?  So  far  as  we  know  from 
past  experiences,  antilytic  substances  in  serum  may  be  either  anti- 
amboceptors  or  anticomplements.  The  data  contained  in  the 
experiments  of  Pfeiffer  and  Friedberger  leave  no  room  for  doubt 
that  antiamboceptors  may  be  excluded;  the  authors,  however, 
also  declare  their  disbelief  in  anticomplements  as  the  cause  of  the 
antilytic  action,  and  feel  themselves  compelled  to  postulate  the 
existence  of  new,  hitherto  unknown  substances. 

We  have  carefully  studied  the  experiments  reported  and  believe 
that  two  possible  explanations  present  themselves.  Thus  we  may 
believe  that  the  antilysins  in  question  are  anticomplements,  which 
in  the  native  serum,  are  covered,  i.e.,  hidden,  by  normal  serum 
constitutents.  In  the  digestion  with  bacteria,  these  normal  con- 
stituents are  removed  (amboceptors) .  The  other  possibility  is 
that  through  the  treatment  with  bacteria  the  bacterial  receptors 
are  liberated  in  the  serum  and  there  functionate  as  antiamboceptors. 
This  has  already  been  suggested  by  Besredka 3.  It  is  obvious  that 

1  Reprinted  from  Deutsche  med.  Wochenschrift,  No.  18,  1905. 

2  Pfeiffer  and  Friedberger,  Deutsche  med.  Wochenschr.,  No.  1,  1905. 

3  Besredka,  Bulletin  Pasteur,  T.  iii,  1905. 

601 


602  COLLECTED  STUDIES  IN   IMMUNITY. 

the  second  of  these  two  alternatives  would  at  once  explain  the 
specific  action  of  the  antilysins.  On  the  other  hand,  it  is  difficult 
to  reconcile  it  with  the  findings  of  Pfeiffer  and  Friedberger,  namely, 
"  that  it  is  possible,  out  of  a  mixture  of  inhibiting  serum  and  immune 
serum,  to  extract  the  amboceptor  by  the  subsequent  addition 
of  bacteria,"  While  thus  compelled  to  leave  open  the  interpreta- 
tion of  the  results  reported  by  Pfeiffer  and  Friedberger,  we  should 
like  to  report  on  analogous  findings  which  we  encountered  with 
haBmolytic  sera  in  the  course  of  experiments  made  to  check  up 
Pfeiffer  and  Friedberger's  results.  Owing  to  greater  ease  with 
which  test-tube  experiments  can  be  controlled,  these  experiments 
proved  more  susceptible  to  analysis.  The  bloods  employed  were 
from  sheep  and  pig,  and  these  were  haBmolyzed  by  the  correspond- 
ing immune  sera1  with  guinea  pig  serum  as  complement. 

Neither  combination  is  inhibited  by  inactive  normal  rabbit 
serum,  and  yet,  as  soon  as  this  serum  is  digested  with  sheep  blood 
or  with  pig  blood,  it  is  found  to  have  acquired  antilytic  properties. 
This  inhibition  of  hasmolysis,  moreover,  is  specific,  so  that  when 
sheep  blood-cells  have  been  used  for  treating  the  serum,  the  inhibi- 
tion extends  only  to  the  haemolysis  of  sheep  blood,  but  not  to  that 
of  pig  blood,  and  when  pig  blood  is  used,  the  inhibition  applies 
only  to  pig  blood  homely  sis. 

This  is  illustrated  by  the  following  experiment:  To  10  cc.  inactive 
rabbit  serum  were  added  the  sedimented  c^lls  from  10  cc.  sheep 
(or  pig)  blood;  the  mixture  was  kept  at  37°  C.  for  one  hour,  and 
then  centrigufed  to  separate  the  serum  from  the  blood-cells.  The 
supernatant  fluids  thus  obtained  were  added  in  decreasing  amounts 
to  constant  quantities  (0.1)  of  active  guinea-pig  serum,  and  digested 
for  half  an  hour;  then  1  cc.  of  a  5%  suspension  of  blood  and  a  suitable 
amount  (1J  amboceptor  units)  of  amboceptor  was  added.  Native 
rabbit  serum  was  treated  in  exactly  the  same  manner  as  the  super- 
natant fluids. 

The  following  table  shows  the  degree  of  solution  noted  in  the 
different  combinations.  The  tubes  in  Column  A  contained  sheep 
blood  plus  0.01  cc.  of  the  corresponding  immune  serum;  the  tubes 

1  The  immune  serum  for  the  pig  blood  was  obtained  by  immunizing  a  rabbit 
with  pig  blood;  that  for  sheep  blood  was  obtained  by  immunizing  a  rabbit 
with  ox  blood,  as  this  was  found  by  Ehrlich  and  Morgenroth  (Berlin,  klin. 
Wochen.,  1901,  Nos.  21  and  22)  to  be  hsemolytic  also  for  sheep  blood. 


JOINT  ACTION  OF  AMBOCEPTORS  IN  HAEMOLYSIS. 


603 


in  Column  B,  contained  pig  blood  plus  0.015  of  the  specific  ambo- 
eeptor.     The  figures  in  each  column  denote: 

1.  Native  rabbit  serum. 

2.  Rabbit  serum  treated  with  sheep  blood. 

3.  Rabbit  serum  treated  with  pig  blood. 


TABLE   I. 


Amount  of 

A 

B 

Rabbit 

Serum. 

cc. 

1 

a 

3 

1 

2 

3 

1.0 

0 

0 

0.5 

0 

0 

0.25 

0 

0 

0.15 
0.1 

3 
S 

ft.  trace 
slight 

1 

1 

3 

0> 

0 
faint  trace 

0.05 
0.025 
0.015 

[  » 

o 

slight 
almost  complete 
complete 

& 

1 

Q 

'ft 

8 

"ft 

I 

slight 
moderate 
strong 

0.01 

strong 

0.0 

• 

- 

complete 

This  table  gives  a  beautiful  illustration  of  the  point  noted  by 
Pfeiffer  and  Friedberger,  namely,  that  the  rabbit  serum,  which 
has  no  antilytic  properties  whatever,  exerts  a  specific  antilytic 
action  after  it  has  been  treated  with  the  corresponding  blood-cells. 
It  is  a  simple  matter  to  show  that  this  antilytic  action  is  not  directed 
against  the  amboceptors.  One  need  merely  mix  amboceptor  and 
inhibiting  serum,  and  then  digest  the  blood  cells  in  this  mixture. 
After  centrifuging,  it  will  be  found  that  the  sedimented  blood-cells 
are  readily  hsemolyzed  on  the  addition  of  complement.  This, 
of  course,  shows  that  the  amboceptor  cannot  have  been  affected. 
Under  these  circumstances,  and  in  the  light  of  our  past  experiences, 
we  would  ascribe  the  action  to  anticomplements,  but  in  doing  so 
we  encounter  apparently  a  great  difficulty,  the  specificity  of  action. 
But  is  this  specificity  really  irreconcilable  with  the  assumption  of 
an  anticomplement  action?  It  seems  to  me  that  no  such  difficulties 
exist  in  our  case,  and  would  ask  the  reader's  attention  to  the  fol- 
lowing considerations : 

It  can  be  shown  that  the  inhibiting  effect  produced  by  a  serum 
after  digestion  with  a  particular  species  of  blood  (in  our  case  with 
sheep  blood),  is  due  essentially  to  the  absorption  of  normal  ambo- 


604 


COLLECTED   STUDIES   IN   IMMUNITY. 


ceptors  acting  on  sheep  blood-cells.  If  one  allows  the  normal 
amboceptors  to  participate  in  the  reaction  by  themselves,  it  will  be 
found  that  the  antilytic  effect  is  not  produced. 

The  demonstration  is  made  as  follows:  An  inhibiting  serum, 
prepared  by  treating  rabbit  serum  with  sheep  blood-cells,  is  mixed 
with  complement  (0.1  cc.  guinea-pig  serum)  and  allowed  to  act 
on  sheep  blood-cells  which  have  been  sensitized  in  one  case  with 
immune  serum,  in  another  case  with  this  and  normal  rabbit  serum. 
The  result  is  shown  in  the  following  table. 

In  Column  I  the  reagent  consisted  always  of  1  cc.  5%  sheep 
blood  sensitized  with  0.002  cc.  immune  serum  obtained  by  im- 
munizing a  rabbit  with  ox  blood-cells. 

In  Column  II,  the  sheep  blood  was  treated  in  exactly  the  same 
manner,  and  then  digested  with  0.5  cc.  normal  rabbit  serum,  where- 
upon the  blood  was  freed  from  serum  by  centrifugalization. 


TABLE  II. 


Amount  of  Rabbit 

Serum  Previously 
Treated  with 

I 

II 

Sheep  Blood-cells. 

cc. 

1.0 

0 

0.5 

0 

0.25 
0.15 

0 
faint  trace 

complete 

0.1 

slight 

0.0 

complete 

It  will  be  seen  from  the  table,  that  through  the  coaction  of 
the  normal  amboceptors  of  rabbit  serum,  the  antilytic  action  dis- 
appears, and  this  at  once  explains  why  the  inhibiting  function 
should  be  absent  in  native  serum.  The  inhibiting  antibodies  are 
really  present  in  native  rabbit  serum  from  the  outset,  but  they 
are  hidden  by  the  simultaneous  action  of  the  normal  amboceptors. 
The  experiment  further  shows  that  the  digestion  of  serum  with 
blood-cells  does  not  bring  about,  for  example,  a  tearing  off  of  receptors 
through  the  agency  of  the  normal  amboceptors.  (Such  a  combina- 
tion in  the  serum  fluid,  moreover,  would  really  act  like  an  anti- 
complement).  Column  II  shows  that  the  normal  amboceptors  are 
really  bound  by  the  blood-cells.  From  the  behavior  of  the  various 


JOINT  ACTION  OF  AMBOCEPTORS  IN  H^MOLYSIS.          605 

combinations,  we  must  furthermore  conclude  that  the  absence 
of  antilytic  action  of  native  serum  is  only  apparent.  Haemolysis 
of  the  sheep  blood-cells  by  the  immune  serum  is  inhibited,  but  in 
place  of  this  the  normal  amboceptors  of  rabbit  serum  come  into 
play  and  effect  haemolysis  with  the  aid  of  the  complement  of  guinea- 
pig  serum.  This,  of  course,  affords  a  natural  explanation  for  the 
specificity  of  the  phenomenon.  The  rabbit  serum  which  was  treated 
with  sheep  blood-cells  has  lost  the  amboceptors  for  sheep  blood, 
but  still  contains  those  for  pig  blood.  Hence  it  inhibits  only  the 
haemolysis  of  sheep  blood  by  immune  serum.  When  the  serum 
is  treated  with  pig  blood,  the  behavior,  of  course,  is  just  the  reverse 
of  this. 

This  explanation  of  the  specificity  harmonizes  very  well  with 
the  view  that  the  inhibiting  substances  are  anticomplements.  It 
is  only  necessary  to  assume  that  the  anticomplements  act  specifically 
in  the  sense  that  under  suitable  conditions  only  the  immune  and 
not  the  normal  amboceptors  are  prevented  from  combining  with 
the  complement.  It  might  be  assumed,  for  example,  that  the 
activation  of  normal  and  immune  amboceptors  is  effected  by  dif- 
ferent complements.  It  seems  simpler,  however,  to  assume  that 
the  complementophile  group  of  the  normal  amboceptors  has  a 
greater  affinity  than  that  of  the  immune  amboceptor.  At  first 
sight  this  may  appear  remarkable,  but  it  is  not  really  so.  It  is 
true  that  one  can  usually  regard  the  immune  amboceptors  as  having 
the  stronger  affinity,  but  this  greater  affinity  applies  only  to  the 
cytophile  group,  i.e.,  the  group  whose  occupation  really  gave  rise 
to  the  immunity  reaction.  So  far  as  the  normal  amboceptors  are 
concerned,  there  is  another  reason  for  believing  that  the  comple- 
mentophile apparatus  possesses  a  greater  affinity.  Anticomple- 
ments, as  Ehrlich  and  Morgenroth  1  have  already  shown,  are  noth- 
ing more  than  amboceptors  which  have  reached  the  blood  stream. 
According  to  this  view,  artificially  produced  anticomplements 
are  amboceptors  which  differ  from  the  amboceptors  produced  in 
response  to  cells  injections,  only  in  the  fact  that  their  thrusting- 
off  is  due  to  the  occupation  of  their  complementophile  group.  Orig- 
inating in  this  way,  a  natural  selection  of  complementophile  groups 
with  the  greatest  affinity,  of  course,  occurs  and  this  subsequently 
shows  itself  in  the  increased  affinity  for  the  anticomplements. 

1  Ehrlich  and  Morgenroth.  Fifth  Communication  on  Haemolysins.  See 
page  71  of  this  volume. 


606  COLLECTED   STUDIES  IN  IMMUNITY. 

Since  experience  has  shown  that  normal  sera  so  frequently 
exert  anticomplementary  powers,  we  are  compelled  to  assume 
that  the  normal  amboceptors,  of  which,  as  is  well  known,  large 
numbers  circulate  in  the  blood,  generally  possess  a  high  affinity  to  the 
complement.  Thanks  to  this  high  affinity  they  are  able  to  deflect 
the  complement  from  the  amboceptor  concerned  in  the  reaction. 
Naturally,  the  question  whether  in  a  given  case  the  amboceptor 
is  to  act  as  such  or  as  anticomplement,  will  depend  in  general  on 
whether  it  fits  the  given  species  of  cell  or  not.  In  any  event,  the 
anticomplementary  action  as  thus  conceived  corresponds  entirely 
to  Neisser  and  Wechsberg's  phenomenon  of  deflection  of  comple- 
ment by  an  excess  of  amboceptor, 

Returning  now  to  the  problem  under  discussion,  we  find  that 
this  finds  a  ready  explanation  along  the  lines  indicated.  This 
will  be  clear  on  studying  the  schematic  figure  appended. 


FIG.  1.  FIG.  2. 

I A  =  immune  amboceptor.     NA  =  normal  amboceptor.     AC  =  normal  ambocep- 
tors  functioning  as  anticomplements.     C  =  complement. 

In  Fig.  1  is  represented  the  action  of  native  rabbit  serum  on 
the  ha3molysis  of  sensitized  sheep  blood-cells  by  guinea-pig  com- 
plement. The  sheep  blood-cells  are  loaded  with  the  immune  ambo- 
ceptor (I A).  The  normal  amboceptor  of  rabbit  serum  fitting  to 
sheep  blood-cells  (NA)  has  likewise  been  anchored  by  the  cell,  and 
has  laid  hold  of  the  complement.1 

In  Fig.  2  the  normal  rabbit  serum,  through  digestion  with  sheep 
blood-cells,  has  lost  its  amboceptor  for  these  cells.  Under  these 

1  The  higher  affinity  of  the  normal  amboceptors  will  be  still  further  increased 
in  favor  of  those  bound  to  the  cell,  for  it  is  well  known  that  in  combining  with 
the  cell  the  complementophile  group  acquires  an  increased  affinity. 


JOINT  ACTION  OF  AMBOCEPTORS  IN  HAEMOLYSIS.         607 

circumstances  the  free  normal  amboceptors,  which  act  as  anti- 
complements  (AC)  come  into  play  and  deflect  the  complement 
form  the  immune  amboceptor  (I A}. 

It  is  understood,  of  course,  that  in  addition  to  these  changes 
in  affinity,  some  significance  must  also  be  attached  to  the  law  of 
mass  action.  Thus,  if  a  very  small  quantity  of  normal  ambo- 
ceptors united  to  cells  is  placed  beside  an  enormous  number  of 
free  anticomplements,  it  is  possible  that  a  deflection  of  complement 
may  occur.  In  dealing  with  native  normal  sera,  such  a  dispropor- 
tion is  out  of  the  question,  for  by  increasing  the  quantity  of  anti- 
complements  there  is  also  an  increase  in  the  amboceptors  fitting 
the  cell.  On  the  other  hand,  if  the  blood-cells  have  only  been 
slightly  sensitized  and  when  then  large  amounts  of  the  inhibiting 
serum  are  employed,  a  slight  antilytic  effect  may  be  produced. 
If  due  regard  is  given  to  the  relative  amounts  of  the  factors,  and  the 
blood-cells  are  sensitized  with  the  proper  proportion  of  normal 
serum,  no  trouble  will  be  experienced  in  observing  the  absence 
of  antilytic  action  against  the  normal  amboceptor.  For  the  sake 
of  completeness  the  following  experiment  is  appended. 

Two  series  of  test  tubes  are  prepared,  the  first  containing  0.1 
cc.  guinea-pig  serum  plus  decreasing  amounts  of  native  rabbit 
serum,  the  other  containing  the  same  amount  of  guinea-pig  serum 
plus  decreasing  amounts  of  rabbit  serum  which  has  previously  been 
absorbed  with  sheep  blood-cells.  To  each  of  the  tubes  is  added  then 
1  cc.  5%  sheep  blood-cells  which  have  previously  been  sentisized 
with  0.5  cc.  normal  rabbit  serum,  and  separated  from  the  serum 
by  centrifugalization.  The  result  is  shown  in  the  following  table. 
The  control  with  immune  amboceptor  is  shown  in  Column  I,  Table  II. 


TABLE   III. 


Amount  of  Rabbit 
Serum, 
cc. 

A. 

Native  Rabbit 
Serum. 

B. 

Rabbit  Serum 
Absorbed  with 
Sheep  Blood. 

1.0 

0.5 

0.25 
0.15 

complete 

complete 

0.1 

0 

608  COLLECTED   STUDIES   IN   IMMUNITY. 

A  number  of  observations  made  during  the  course  of  other  experi- 
ments gives  additional  support  to  the  view  that  the  inhibiting 
action  is  due  to  anticomplements  whose  action  is  hidden,  in  native 
sreum,  by  the  normal  amboceptors.  Thus,  it  was  possible  to  bring 
about  inhibition  by  absorption,  only  when  the  serum  employed 
already  contained  amboceptors  for  the  blood-cells  in  question. 
Where  these-  amboceptors  were  absent,  no  change  whatever  was 
produced  by  the  absorption,  the  serum  either  inhibiting  equally 
well  before  and  after  absorption,  or  not  inhibiting  at  all.  Normal 
rabbit  serum,  for  example,  is  in  no  way  changed  when  absorbed 
with  ox  blood-cells,  because  it  lacks  fitting  receptors  for  these  cells. 
Owing  to  the  fact,  however,  that  it  contains  anticomplements, 
rabbit  serum  even  in  its  native  state  exerts  an  antilytic  effect  on 
ox-blood  haemolysis,  and  this  action  is  unaffected  by  absorption 
either  with  ox  blood  or  sheep  blood.  In  this  connection  the  indi- 
vidual variations  observed  in  the  behavior  of  rabbit  sera  toward 
sheep  blood  is  most  instructive.  Thus,  I  have  encountered  rabbit 
sera  in  which,  by  chance,  the  amboceptors  for  sheep  blood  were 
practically  absent.  These  sera,  however,  even  in  the  native  state, 
possessed  an  antilytic  effect  on  sheep-blood  haemolysis,  and  this 
was  unaffected  by  treatment  with  sheep  blood-cells. 

Finally  mention  should  be  made  of  a  circumstance  which  makes 
it  highly  probable  that  the  substances  in  question  are  anticomple- 
ments. We  have  seen  that  the  inhibiting  serum  produces  its  effect 
when  guinea-pig  serum  is  used  as  complement.  On  the  other  hand, 
no  inhibition  will  be  produced  if  rabbit  serum  is  used  as  comple- 
ment. The  amboceptors  present  are  complemented  with  rabbit 
serum  just  as  well  as  with  guinea-pig  serum,  and  the  failure  of 
absorbed  rabbit  serum  to  inhibit  when  rabbit  serum  is  used  as  com- 
plement can  be  readily  understood  if  we  regard  the  inhibition  as 
due  to  anticomplements  as  already  set  forth,  for  it  is  well  known 
that  autoanticomplements  are  uncommon. 

It  is,  of  course,  impossible  for  us  to  say  whether  the  data  here 
reported  are  applicable  to  the  observations  made  by  Pfeiffer  and 
Friedberger  on  bacteria.  From  what  has  been  said  it  is  apparent 
that  the  specificity  observed  by  those  authors  would  agree  very 
well  with  the  anticomplemenjb  hypothesis.  Nor  is  this  hypothesis 
contradicted  by  the  fact  that  a  certain  excess  of  amboceptor  nullifies 
the  paralyzing  action  of  the  inhibiting  serum.  In  anticomple- 
ment  actions  the  quantitative  relations  between  amboceptor, 


JOINT  ACTION  OF  AMBOCEPTORS  IN  HAEMOLYSIS.        609 

complement,  and  anticomplement  are  so  important  that  the  failure 
of  inhibition  when  large  amounts  of  amboceptor  are  employed 
may  be  ascribed  to  the  disproportion  between  the  reacting  sub- 
stances. This  phase  of  the  subject  has  been  investigated  by  Mor- 
genroth  and  Sachs.1  Finally  mention  should  be  made  of  a  fact 
reported  by  Pfeiffer  and  Friedberger  which  strongly  supports  the 
anticomplement  theory.  By  means  of  bacterial  absorption  they 
succeeded  in  converting  normal  rabbit,  goat,  and  pigeon  serum 
into  inhibiting  serum,  but  failed  to  convert  giunea-pig  serum.  Yet 
it  is  well  established  by  experiments  in  this  direction  that  bacter- 
iolysis takes  place  in  the  peritoneal  cavity  of  guinea  pigs,  that, 
in  other  words,  these  animals  do  furnish  complement.  The  negative 
result  obtained  with  guinea-pig  serum,  therefore,  may  be  regarded 
as  indicating  the  absence  of  autoanticomplements,  and  the  exper- 
iment affords  additional  reason  for  believing  that  the  antagonistic 
aubstances  observed  by  Pfeiffer  and  Friedberger  are  probably 
anticomplements. 

1  Morgenroth  and  Sachs.     Berliner  klin.  Wochenschrift,  No.  35,  1902. 


XLV.    THE   POWER   OF  NORMAL  SERUM  TO  DEFLECT 

COMPLEMENT.1 

By  Dr.  HANS  SACHS,  Member  of  the  Institute. 

IN  a  previous  paper2  the  writer  discussed  the  action  of  certain 
substances  in  normal  serum  which,  according  to  Pfeiffer  and  Fried- 
berger,3 exerted  antibacteriolytic  effects.  A  recent  study  by  Gay  4 
leads  me  to  take  up  the  subject  anew.  Pfeiffer  and  Friedberger 
had  shown  that  normal  sera  which  by  themselves  possessed  no 
antibacteriolytic  properties,  acquired  such  properties  if  they  were 
previously  digested  with  bacteria.  Moreover  the  sera  obtained 
by  this  treatment  exert  specific  antilytic  properties,  that  is  to  say, 
a  serum  digested  with  cholera  vibrios  protects  only  chlorea  vibrios 
against  bacteriolysis,  etc.  I  thereupon  studied  the  same  condi- 
tions by  means  of  hcemolytic  test-tube  experiments,  and  was  able 
to  confirm  the  author's  findings.  Rabbit  serum  digested  with 
sheep  blood-cells  exerts  antihaemolytic  effects  directed  practically 
entirely  against  the  haemolysis  of  sheep  blood.  My  conception 
of  the  mechanism  of  this  action  differs  from  that  of  Pfeiffer  and 
Friedberger  only  in  that  I  do  not  regard  the  inhibiting  substances 
concerned  as  new,  hitherto  unknown  bodies.  I  believe  that  this 
inhibiting  effect,  at  least  in  the  case  of  hsemolysins,  is  due  to  ambo- 
ceptors,  acting,  as  they  often  do,  like  anticomplements.  That 
such  amboceptors  occur  in  normal  serum  is  well  known  from  nu- 
merous observations.  At  any  rate,  the  views  of  Pfeiffer  and  Fried- 
berger and  my  own  probably  agree  in  regarding  the  inhibiting 
substances  in  the  serum  as  preformed,  their  action  in  native  serum 


1  Reprinted  from  Centralblatt  f.  Bacteriologie,  Vol.  XL,  1906. 

2  Sachs.     Deutsche  med.  Wochenschrift,  No.  18,  1905. 

3  Pfeiffer  and  Freidberger,  ibid.  No.  1,  and  also  No.  29,  1905. 

4  Gay,  Centralblatt  Bacteriologie,  Orig.  XXXIX,  1905.     See  also  Bordet- 
Gay,  Collected  Studies,  Wiley  &  Sons,  1909. 

610 


POWER  OF  NORMAL  SERUM  TO  DEFLECT  COMPLEMENT.     611 

being  hidden  by  the  normal  amboceptors  which  are  removed  by 
the  digestion  with  blood-cells  or  bacteria. 

Gay  believes  otherwise.  He  has  repeated  my  experiments, 
especially  those  dealing  with  haBmolysis,  and  concludes  that  my 
explanation  is  "certainly  incorrect."  Gay  believes  that  the  cause 
of  the  phenomenon  described  is  to  be  sought  in  a  binding  of  com- 
plement by  precipitates.  According  to  him  the  precipitin  is  in  the 
sheep-blood  immune  serum;  the  precipitable  substance  is  in  the 
rabbit  serum  digested  with  sheep  b  ood  and  comes  from  traces  of 
serum  remaining  on  the  sheep  blood  after  insufficient  washing. 
This  explanation,  at  first  sight,  seems  most  reasonable.  We  know 
from  the  researches  of  Gengou l  that  the  combination  resulting 
from  the  union  of  serum  albumin  and  a  corresponding  antiserum 
has  the  power  to  bind  complement.  Through  the  recent  investiga- 
tions of  Moreschi 2  and  of.  Gay 3  a  great  deal  of  interest  has  been 
aroused  in  this  property,  and  M.  Neisser  and  1 4  have  reported  on 
experimental  studies  in  which  we  sought  to  utilize  the  complement- 
binding  power  of  albuminous  bodies  laden  with  antiserum  in  a 
forensic  blood  test.  For  the  question  here  at  issue  it  matters  not 
whether  the  precipitate  as  such  absorbs  the  complement,  or  whether, 
as  we  believe,  the  albuminous  bodies  are  sensitized  by  specific 
amboceptors  in  Gengou's  sense,  so  that  they  then  bind  the  com- 
plement just  as  do  sensitized  cells.  The  main  point  is  that  accord- 
ing to  Gay's  view  the  inhibition  of  hsmolysis  must  be  due  to  the 
interaction  of  sheep  serum  and  the  immune  serum  acting  on  sheep 
blood. 

The  experiments  made  by  Gay  apparently  corroborate  his  assump- 
tion. Thus  when  the  sheep-blood  corpuscles  used  for  treating  the 
rabbit  serum  were  washed  five  successive  times  with  physiological 
salt  solution,  he  found  that  the  centrifuged  rabbit  serum  no  longer 
produced  inhibition,  whereas  when  the  serum  was  treated  with 
sheep  blood  washed  but  once,  it  produced  the  inhibition  which 
I  had  described.  The  difference  which  I  observed  in  the  behavior 
of  normal  and  immune  amboceptors  of  rabbit  serum,  so  far  as 
the  inhibiting  action  of  rabbit  serum  treated  with  sheep  blood 
is  concerned,  Gay  believes,  is  only  an  apparent  one.  Normal 

1  Gengou,  Annales  Pasteur,  Tome  XVI,  1902. 

2  Moreschi,  Berliner  klin.  Wochensehrift,  No.  37,  1905. 

3  Gay,  Centralblatt  Bacteriologie,  Orig.  XXXIX,  1905. 

4  Neisser,  M.,  and  Sachs,  Berliner  klin.  Wochensehrift,  No.  44,  1905. 


612  COLLECTED  STUDIES   IN  IMMUNITY. 

serum  simply  does  not  contain  the  antibodies  (precipitings)  acting 
on  the  sheep  serum,  and  this  is  why  there  is  no  inhibition.  In 
one  experiment,  however,  I  called  attention  to  the  fact  that  the 
haemolysis  of  blood  cells  sensitized  only  with  immune  serum  is 
prevented  by  the  inhibiting  serum,  whereas  blood-cells  sensitized 
with  immune  serum  and  then  also  with  normal  serum  are  dissolved 
under  these  conditions.  In  both  cases  after  the  amboceptors  had 
been  anchored  I  separated  the  serum  fluid  by  centrifuging,  It 
so  happened  that  I  expressed  myself  somewhat  differently  in  the 
second  case,  and  this  has  led  to  a  misconception.  In  the  second 
case  I  said  "the  blood-cells  were  digested  with  serum  and  then 
freed  from  serum  fluid  by  centrifuging  " ;  in  the  first  case  I  merely 
said  "the  reagent  used  was  sheep  blood  sensitized  with  immune 
serum."  By  "sensitized  blood,"  of  course,  I  mean  blood-cells 
which,  after  treatment  with  amboceptors,  are  separated  by  centrifuge. 
In  fact,  in  another  experiment  contained  in  this  study  I  expressly 
state  "sheep  blood +immune  serum."  However,  I  am  willing  to 
admit  that  my  mode  of  expression  might  give  rise  to  doubts.  Gay, 
however,  seems  to  know  my  experimental  technique  better  than 
even  I  myself.  He  declares  simply  that  I  had  centrifuged  in  the 
second  case,  i.e.,  had  removed  the  precipitating  portion  of  the 
immune  serum,  and  had  not  done  so  in  the  first  case.  My  experi- 
ments therefore  contained  "  a  grave  experimental  error."  Through 
his  own  experiments,  Gay  believes  to  have  furnished  "a  complete 
refutation  of  Sach's  hypothesis." 

Gay  has  made  a  regrettable  mistake.  Moreover,  in  repeating 
my  experiments  he  has  allowed  a  grave  error  to  creep  into  his  own 
technique.  It  really  is  immaterial,  in  my  experiments,  whether  the 
immune  serum  is  centrifuged  from  the  blood-cells  or  not,  since  the 
immune  serum  I  employ  has  so  high  an  amboceptor  content  that  the 
quantity  used  for  sensitizing  (0.002  cc.)  is  only  about  1-200  of  that 
employed  by  Gay.  According  to  my  experience,  this  quantity  is 
too  small  to  effect  a  precipitation  or  sensitization  of  the  albuminous 
bodies  of  the  serum.  Nevertheless  I  have  made  a  number  of  exper- 
iments with  my  immune  serum  according  to  the  procedure  outlined 
by  Gay.  I  treated  rabbit  serum  with  sheep  blood  washed  once, 
and  also  with  sheep  blood  washed  five  times.  Both  lots  of  serum 
so  treated  proved  equally  antihaemolytic,  whereas  native  rabbit 
serum  possessed  no  inhibiting  action  whatever.  This  is  illustrated 
by  the  following  protocol. 


POWER  OF  NORMAL  SERUM  TO  DEFLECT  COMPLEMENT.     613 


Decreasing  amounts  of  inactive  rabbit  serum  are  digested  with  0.05  cc. 
guinea-pig  serum.  Then  1  cc.  5%  sheep  blood  washed  five  times  plus  0.0015  cc. 
amboceptor  (serum  from  a  rabbit  immunized  with  ox  blood)  are  added.  In 
the  following  table 

A  denotes  native  rabbit  serum; 

B  rabbit  serum  treated  with  sheep  blood  washed  once; 

C  rabbit  serum  treated  with  sheep  blood  washed  five  times. 


Amounts  of 

Degree  of  Haemolysis. 

Rabbit  Serum. 

cc. 

A 

B 

C 

1.0 

1 

0 

0 

0.5 

0 

0 

0.25 
0.15 

>•  complete 

0 
moderate 

0 
moderate 

0.1 

1  1 

" 

0 

complete 

complete 

The  experiment  by  which  Gay  seeks  to  explain  my  results  is 
therefore  entirely  valueless  so  far  as  my  experiment  is  concerned. 
To  be  sure,  Gay  Helieves  to  have  followed  my  technique  exactly, 
yet  in  one  important  point  he  has  not  done  so.  The  hsemolytic 
immune  serum  with  which  he  worked  was  markedly  weak,  the 
solvent  dose  being  0.2  cc.,.  while  0.001  cc.  of  my  serum  still  brought 
about  complete  solution.  Gay  employed  0.4  cc.  immune  serum, 
while  I  used  but  0.002  cc.,  i.e.,  1-200  of  his  dose.  It  is  very  well 
possible  that  the  antibodies  which  sensitize  serum  albuminous 
bodies,  the  precipitating  substances  as  Gay  believes,  are  present 
in  0.4  cc.  immune  se'rum;  in  amounts  as  small  as  0.002  cc.  they 
are  almost  certainly  absent,  and  I  never  observed  any  precipitin 
action  with  these  amounts.  It  is  evident,  therefore,  that  with  the 
large  doses  of  immune  serum  employed  by  Gay  the  presence  of 
slight  amounts  of  sheep  serum  might  well  make  a  difference  in  the 
result,  while  this  would  be  a  negligible  factor  in  my  experiments. 

But  how  are  we  to  explain  the  fact  that  Gay  after  treating  the 
rabbit  serum  with  sheep  blood  which  had  been  washed  five  times 
was  unable  to  demonstrate  the  inhibiting  action  described  by  me? 
This  again  is  due  to  the  unfortunate  employment  of  the  weak  immune 
serum.  Gay  has  evidently  been  working  with  normal  amboceptors. 
It  is  well  known  that  rabbit  serum  normally  dissolves  sheep  blood, 
and  the  ordinary  strength  of  this  hamolytic  power  corresponds 


614  COLLECTED  STUDIES   IN   IMMUNITY. 

entirely  to  that  of  Gay's  immune  serrum.  The  complete  solvent 
dose  of  normal  rabbit  serum  fluctuates  in  most  instances  between 
0.25  and  0.1  cc.  The  solvent  dose  of  Gay's  immune  serum  was 
0.2  cc.  The  quantity  employed  by  Gay,  0.4  cc.,  probably  suffices 
with  any  rabbit  serum  to  completely  dissolve  Ice.  5%  .sheep  blood 
on  the  addition  of  0.1  cc.  guinea-pig  serum.  Hence  it  is  not  at  all 
impossible  that  Gay  employed  a  serum  which  contained  no  immune 
amboceptors  whatever,  and  represented  merely  an  albumin  anti- 
serum.  If  amboceptors  were  artificially  produced,  they  were 
certainly  present  in  so  small  concentration  as  to  be  unable  to  in- 
crease the  action  of  the  normal  amboceptors  to  any  appreciable 
degree.  In  my  paper,  however,  I  distinctly  stated  that  the  inhibit- 
ing sera  obtained  by  treatment  with  blood-cells  acted  only  against 
haBomlysis  produced  by  immune  amboceptors,  and  that  this  anti- 
lytic  action  was  prevented  by  the  action  of  the  normal  amboceptors. 
Just  this  constituted  my  explanation  for  the  absence  of  the  inhibit- 
ing function  in  native  serum,  for  I  assumed  that  the  inhibiting 
antibodies  were  already  present  in  native  rabbit  serum  and  were 
merely  hidden  by  the  simultaneous  action  of  the  specific  normal 
amboceptors. 

Gay's  experiments  thus  constitute  an  involuntary  complete  con- 
fimation  of  my  views,  and  it  is  to  be  regretted  that  Gay  has  allowed 
himself  to  be  so  misled  in  the  interpretation  of  my  experiments.  The 
incorrectness  of  his  views  should  have  struck  him  from  a  number 
of  statements  in  my  first  paper.  If  his  idea  was  correct  it  follows 
that  the  inhibiting  action  should  appear  in  every  rabbit  serum 
treated  with  insufficiently  washed  sheep  or  ox  blood.  I  distinctly 
stated,  however,  that  I  could  only  then  demonstrate  an  inhibiting 
effect  through  absorption  with  blood-cells  when  the  serum  under 
examination  from  the  outset  contained  amboceptors  for  the  species 
of  blood  in  question.  I  said  particularly  that  normal  rabbit  serum, 
which  contains  no  amboceptors  for  ox  blood,  is  in  no  wise  changed 
by  absorption  with  ox  blood,  i.e.,  it  neither  before  nor  after  treatment 
with  ox  blood  does  it  inhibit  ha3molysis  of  sheep  blood  by  immune 
serum,  while  on  the  ha3molysis  of  ox  blood  it  exerts  the  same  inhibit- 
ing power  before  and  after  treatment.  Finally  I  called  attention 
to  a  number  of  rabbit  sera  in  which,  quite  exceptionally,  the  ambo- 
ceptors for  sheep  blood  were  absent.  These  sera  from  the  outset 
were  antilytic  for  the  haemolysis  of  sheep  blood,  and  they  remained 
so  in  unaltered  degree  after  digestion  with  sheep  blood.  After 


POWER  OF  NORMAL  SERUM  TO  DEFLECT  COMPLEMENT.     615 

all  this  it  is  absolutely  necessary  to  conclude  that  the  inhibiting 
substances  are  already  present  in  native  serum,  and  that  their  action 
in  this  serum  is  merely  disguised  by  the  simultaneous  action  of  the 
normal  amboceptors.  If  the  latter  are  removed  by  absorption 
with  blood-cells,  the  antilytic  power  of  the  inhibiting  substances 
becomes  manifest.  Gay's  attempt  to  refute  this  conception  has 
thus  come  to  naught,  and  all  because  of  a  circumstance  in  his  tech- 
nique which  Gay  himself  perhaps  not  unjustly,  would  term  a  "grave 
experimental  error." 


XLVI.    THE  JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS 

IN  HAEMOLYSIS  AND  THEIR  RELATION  TO 

THE  COMPLEMENTS.1 

By  Drs.  H.  SACHS  and  J.  BAUER. 

THERE  is  still  no  unanimity  of  opinion  concerning  the  mechanism 
of  the  cytotoxic  action  of  blood  serum.  Most  of  the  authors,  to 
be  sure,  have  accepted  the  amboceptor  theory  of  Ehrlich  and  Mor- 
genroth.  According  to  this  view,  the  thermostable  components 
of  the  serum  posses  two  haptophore  groups,  one  combining  with 
the  cell  and  the  other  with  the  complement,  the  labile  component 
of  the  serum.  Bordet,  however,  continues  most  ingeniously  to 
defend  an  opposing  view.  In  the  sensitization  theory  advocated 
by  this  distinguished  investigator,  the  existence  of  a  direct  relation- 
ship between  amboceptor  and  complement  is  denied.  Accord- 
ing to  this  view,  which  is  based  on  molecular  adhesion,  the  cell 
is  sensitized  by  the  amboceptor  so  that  it  becomes  vulnerable  to 
the  action  of  the  complement.  So  far  as  can  be  discovered  blood 
cells  (which  constitute  the  ordinary  material  on  which  to  study  the 
mechanism  of  amboceptor  action)  do  not  by  themselves  react  with 
complement,  and  it  has  therefore  been  impossible  to  prove  the  cor- 
rectness of  the  sensitization  theory  experimentally.  The  theory 
can  only  be  defended  indirectly,  by  showing  that  there  is  no  direct 
relation  between  amboceptor  and  complement.  Bordet' s  demon- 
strations have  therefore  been  limited  to  pointing  out  objections 
in  experiments  supporting  the  amboceptor  theory.  It  is  not  our 
intention  to  present  all  the  material  bearing  on  this  point.  One 
of  us2  has  recently  reviewed  the  subject  on  the  ilght  of  our  present 
knowledge.  Suffice  it  to  say  that  the  refutation  of  experiments 

1  Reprinted  from  Arbeiten  u.  d.  kgl.  Institut  f.  experimentelle  Therapie  zu 
Frankfurt  a.  M.     Heft  3,  Jena,  1907. 

2  Sachs,  Die  Hsemolysine  und  die  cytotoxischen  Sera.     Lubarsch-Ostertags 
Ergebnisse  der  Pathologic.     Vol.  11,  1907. 

616 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  617 

which  appeared  clearly  to  indicate  the  direct  union  of  amboceptor 
and  complement  is  not  at  all  sufficient  to  overthrow  the  ambo- 
ceptor theory.  Attacking  our  interpretation  of  a  phenomenon 
which  played  an  important  role  in  proving  the  existence  of  direct 
relations  between  amboceptor  and  complement,  Bordet  and  Gay1 
in  a  recent  paper,  report  an  experiment  which  they  believe  con- 
troverts our  view.  Going  still  further,  these  authors  conclude  that 
the  amboceptor  theory  must  be  abandoned  as  fallacious.  We  fail 
to  see  the  force  of  this  conclusion.  For  even  if  the  proof  adduced 
by  Bordet  and  Gay  in  this  single  instance  were  accepted  as  irre- 
futable, it  would  only  show  that  the  direct  demonstration  of  the 
amboceptor  thoery  is  impossible.  The  authors  have  not  brought 
forward  a  single  fact  which  contradicts  the  amboceptor  theory. 
If,  then,  in  the  following  pages  we  take  up  at  length  the  observa- 
tions of  Bordet  and  Gay,  it  is  not  because  we  consider  it  necessary 
to  renew  the  old  discussion  "  amboceptor  or  substance  sensibilatrice?" 
but  merely  because  of  the  great  interest  of  the  observations.  Further- 
more the  interpretation  given  by  the  authors  is  so  peculiar  that  it 
demands  further  analysis. 


The  case  discussed  by  Bordet  and  Gay  deals  with  a  combina- 
tion previously  described  by  Ehrlich  and  Sachs,2  namely  haemolysis 
of  guinea-pig  blood  through  the  combined  action  of  inactive  ox 
serum  and  active  horse  serum.  Ehrlich  and  Sachs  had  found  that 
guinea-pig  erythrocytes,  which  can  be  dissolved  by  a  mixture  of 
inactive  ox  serum  and  horse  serum,  remain  intact  if  they  are  first 
treated  with  inactive  ox  serum,  and  then,  after  removing  the  ox 
serum,  are  digested  with  horse  serum.  This  showed  that  the  con- 
stituent of  ox  serum  has  not  been  bound  by  the  blood  cells.  It 
was  to  be  assumed  that  this  constituent  represented  the  amboceptor, 
and  Ehrlich  and  Sachs  therefore  rightly  concluded  that  in  this 
case  the  amboceptor  had  not  been  bound  by  the  blood-cells,  that 
it  reacted  with  the  cell  only  after  the  amboceptor  and  complement 
had  combined.  The  same  combination  was  subsequently  studied 


1  Bordet  et  Gay,  Annales  de  1'Institut  Pasteur,  No.  6,  Vol.  XX,  1906. 
2Erhlich  and  Sachs,  Berliner  klin.  Wochenschr.  No.  21,  1902.     See  also 
this  volume,  page  209. 


618  COLLECTED   STUDIES   IN   IMMUNITY. 

by  Klein,1  who  found  that  horse  serum,  through  digestion  with 
guinea-pig  blood,  loses  its  complementing  power  for  the  combina- 
tion "guinea-pig  blood  inactive  ox  serum."  Finding  that  the  horse 
serum  suffered  a  loss  of  its  agglutinin  at  the  same  time,  Klein  ad- 
vanced the  view  that  the  complement  was  destroyed  by  the  pro- 
cess of  deglutination.  This  view  was  combated  by  Browning,2 
who  showed  that  the  complements  of  horse  serum  remain  unaffected 
if  the  guinea-pig  blood-cells  are  digested  with  the  serum  at  low 
temperatures  (0°  C.),  although  optimum  conditions  for  the  agglu- 
tinating action  and  for  the  binding  of  agglutinin  are  thus  presented. 
Browning  believes  that  the  reason  for  the  disappearance  of  com- 
plement through  digestion  at  higher  temperatures,  lies  in  the  fact 
that  horse  serum  contains  amboceptors  for  guinea-pig  blood,  which 
amboceptors  serve  to  bind  the  complement  only  at  higher  tem- 
peratures. That  amboceptors  for  guinea-pig  blood  exist  in  horse 
serum  was  demonstrated  by  Morgenroth  and  Sachs,3  who  show 
that  horse  serum  plus  active  guinea-pig  serum  was  able  to  produce 
haemolysis  of  guinea-pig  blood-cells. 

These  authors  demonstrated  further  that  horse  serum  alone, 
even  in  large  doses,  only  rarely  dissolved  guinea-pig  blood-cells, 
This  showed  that  horse  serum  usually  did  not  contain  the  suitable 
" dominant"  complement  fitting  its  own  amboceptor  for  guinea- 
pig  blood.  It  is  well  known  that  an  amboceptor  which  has  been 
anchored  to  a  cell  is  able  to  rob  an  active  serum  of  all  its  comple- 
ment functions.  Furthermore,  according  to  Ehrlich  and  Sachs,4 
under  certain  conditions  even  "non-dominant"  complements 
may  be  anchored  while  "dominant"  complements  remain  in  solu- 
tion. Hence  the  explanation  offered  by  Browning  presented  no 
difficulties.  Browning  assumed  that  the  horse  serum  complement, 
dominant  for  the  ox  amboceptor  but  not  dominant  for  the  horse 
amboceptor,  is  absorbed  by  guinea-pig  blood  through  the  agency 
of  the  serum's  own  amboceptor.  The  loss  of  complement  described 
by  Klein  was  thus  readily  explained  on  the  basis  of  the  ambo- 


1  Klein,  Wiener  klin.  Wochenschr.,  No.  48,  1905. 

2  Browning,  Wiener  klin.  Wochenschr.,  No.  15,  1906. 

3  Morgenroth  and  Sachs,   Berliner  klin.   Wochenschr.,  No.   27,    1902.     See 
also  page  233. 

4  Ehrlich  and  Sachs,  Berliner  kiin.  Wochenschr.  1902,  Nos.  14  and  15.     See 
also  this  volume,  page  195. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  619 

ceptor  theory.  Browning  also  showed  that  a  similar  effect  could 
be  produced  with  other  species  of  blood  which  by  themselves  were 
unable  to  rob  horse  serum  of  its  complement.  It  was  merely  neces- 
sary to  introduce  a  specific  amboceptor.  Ox-blood,  for  example, 
has  no  influence  on  horse  serum.  Nevertheless,  when  treated  with 
a  specific  amboceptor  derived  from  a  rabbit,  it  binds  the  horse 
serum  complement  fitting  inactive  ox  serum,  and  this  binding 
occurs  without  the  prepared  cells  being  dissolved  by  the  horse  serum. 
According  to  BrowTiing,  therefore,  haemolysis  of  guinea-pig  blood 
brought  about  by  the  combined  action  of  inactive  ox  serum  and 
active  horse  serum  is  to  be  explained  as  follows:  The  affinity  pos- 
sessed by  the  ox  amboceptor  for  horse  complement  is  greater  than 
that  possessed  by  the  free  horse  amboceptor.  Haemolysis  occurs 
if  the  ox  serum  and  horse  serum  are  added  at  the  same  time.  If, 
however,  the  horse  serum  is  first  digested  with  guinea-pig  blood, 
the  horse  amboceptor  will  unite  with  the  blood-cell.  This  union 
leads  to  an  increase  in  the  affinity  of  the  complementophile  group 
and  causes  the  complement  to  be  anchored  to  the  horse  amboceptor. 
The  union  between  complement  and  amboceptor  becomes  more 
and  more  firm,  so  that  after  a  tune  not  even  the  ox  amboceptor, 
which  really  possesses  a  higher  affinity  than  the  horse  amboceptor, 
is  able  to  disrupt  the  combination.  (See  figures  1  and  2  of  the 
accompanying  plate.) 

It  is  apparent  that  Bordet  and  Gay  were  unacquainted  with 
the  work  of  Browning.  The  experiments  they  report  are  largely 
identical  with  those  made  by  Klein  and  Browning.  The  follow- 
ing interesting  experiment,  however,  is  entirely  new:  Ox  blood-cells 
loaded  with  amboceptor  do  not  dissolve  in  horse  serum,  but  do  dis- 
solve in  a  mixture  of  active  horse  serum  plus  inactive  ox  serum. 
In  this  case,  the  authors  rightly  reason,  the  ox  serum  cannot  pos- 
sibly act  as  an  amboceptor,  but  must  represent  a  constituent  neces- 
sary for  haemolysis,  but  identical  neither  with  the  amboceptor 
nor  with  the  complement.  Analogously,  in  the  combination  guinea- 
pig  blood  plus  inactive  ox  serum  plus  horse  serum,  the  horse  serum 
is  believed  to  act,  not  as  an  amboceptor,  but  as  a  third  component 
effecting  haemolysis.  Bordet  and  Gay  thus  assume  that  ambo- 
ceptor and  complement  are  present  in  horse  serum  but  are  unable 
to  effect  haemolysis  without  the  presence  of  the  third  component 
present  in  ox  serum.  This  hypothetical  substance  they  term 
"colloide  de  breuf."  According  to  Bordet  and  Gay,  this  colloid 


620  COLLECTED  STUDIES   IN  IMMUNITY. 

has  the  following  properties:  It  is  stable,  resisting  long  standing 
and  heating  to  56°.  It  is  bound  by  the  blood-cells  only  after  these 
have  been  loaded  with  amboceptor  and  complement.  When  so 
bound  it  effects  agglutination  and  haemolysis.  Bordet  and  Gay 
thus  assume  the  existence  of  an  entirely  new  substance  in  horse 
serum,  and  ascribe  to  it  very  important  properties.  The  inter- 
pretation which  these  authors  give  of  the  phenomenon  described 
by  Ehrlich  and  Sachs  is  merely  an  hypothesis  entirely  lacking  in 
proof.  Granted  that  the  role  of  the  ox  serum  in  the  haemolysis 
of  sensitized  ox  blood  by  means  of  horse  serum  cannot  be  looked 
upon  as  an  amboceptor  action,  this  by  no  means  justifies  the  analogous 
conclusion  that  in  the  haemolysis  of  guinea-pig  blood  by  inactive 
ox  serum  and  horse  serum  the  ox  serum  does  not  play  the  part 
of  an  amboceptor.  It  should  at  least  be  shown  that  guinea-pig 
blood  digested  with  horse  serum  (whereby,  according  to  the  view 
of  Bordet  and  Gay,  amboceptor  and  complement  are  bound)  is 
haemolyzed  on  the  subsequent  addition  of  inactive  ox  serum.  Klein 
and  Browning,  however,  showed  that  this  was  not  the  case.  The 
latter,  moreover,  offered  an  explanation  which  harmonized  per- 
fectly with  the  amboceptor  theory.  Bordet  and  Gay  themselves 
failed  when  they  attempted  this  crucial  experiment.  From  the 
fact  that  guinea-pig  blood-cells  which  have  been  treated  with  horse 
serum  are  strongly  agglutinated  by  inactive  ox  serum,  they  con- 
clude, however,  that  a  binding  of  the  "  colloid  "  has  occurred.  We 
should  like  to  point  out  that  haemolysis  and  agglutination  cannot 
be  regarded  as  due  to  one  and  the  same  substance,  and  that  con- 
sequently there  is  no  justification  for  the  conclusion  drawn  by  these 
authors  concerning  the  haemolytic  constituent  of  ox  serum.  Bordet 
and  Gay,  to  be  sure,  seek  to  explain  the  failure  attending  this  impor- 
tant (for  their  conception)  experiment  by  regarding  the  absence 
of  haemolysis  as  due  to  a  marked  antagonistic  effect  exerted  by  the 
strong  agglutination.  They  found  that  such  agglutinated  blood- 
cells  would  not  dissolve  even  when  they  were  resuspended  in  a 
fresh  mixture  of  inactive  ox  serum  and  horse  serum.  We  look  in 
vain,  however,  for  an  experiment  which  would  have  decided  the 
question  absolutely.  Thus,  if  the  guinea-pig  blood-cells  digested 
with  horse  serum  really  do  absorb  the  haemolytic  component  of 
ox  serum,  it  should  be  possible  to  show  that  the  ox  serum  has  lost 
the  power  to  dissolve  guinea-pig  blood  in  conjunction  with  horse 
serum.  Bordet  and  Gay  do  mention  that  inactive  ox  serum  which 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS. 


621 


had  been  treated  with  sensitized  ox  blood  previously  digested  with 
horse  serum  does  dissolve  guinea-pig  blood  in  conjunction  with 
horse  serum  less  rapidly  and  less  actively  than  does  native  (i.e., 
untreated)  ox  serum.  We  should  imagine  that,  according  to  the 
views  of  Bordet  and  Gay,  the  ox  serum  in  this  case  would  have 
been  completely  exhausted.  Be  this  as  it  may,  the  fact  remains 
that  the  one  dicisive  experiment  has  not  been  made. 


II. 

In  our  experiments,  therefore,  we  first  sought  to  fill  this  gap. 
We  made  use  of  5%  suspensions  of  guinea-pig  blood-cells,  which, 
of  course,  were  washed  free  of  serum.  The  ox  serum  was  inact- 
ivated by  half  an  hour's  heating  to  53-54°.  In  all  the  tests  the 
mixtures  were  brought  up  to  the  same  volume  with  physiological 
salt  solution,  and  this  volume  was  never  less  than  2  cc.  nor  more 
than  2.3  to  2.5  cc.  The  titration  of  the  horse  serum  is  shown  in 
the  following  table. 

TABLE  I. 


Haemolysis  of  1  cc.  5%  Guinea-pig  Blood 
by  Means  of  Horse  Serum. 

Amount  of 

Active 

Horse  Serum. 

A 

B 

cc. 

On  the  Addition  of 
0.1  cc.  Inactive 
Ox  Serum. 

Without  any 
Further  Addition. 

0.5 
0.35 

complete 
complete 

' 

0.25 

almost  complete 

0.15 

moderate 

0 

0.1 

little 

0.05 

trace 

0 

0 

After  this  to  each  1  cc.  5%  suspension  guinea-pig  blood  was  added  0.35  cc. 
horse  serum,  i.e.,  sufficient  to  surely  activate,  and  the  mixtures  digested  at 
37°  for  one  hour.  A  test  of  the  decanted  fluids  showed  that  the  active  principle 
had  been  bound  by  the  blood-cells.  (See  Table  II.) 

The  blood  sediments  which  had  thus  been  treated  with  horse  serum  were 
next  digested  for  one  hour  at  37°  with  decreasing  amounts  of  inactive  ox 
serum.  No  haemolysis  occurred.  The  tubes  were  then  centrifuged  and  the 
decanted  fluids  digested  with  0.35  cc.  horse  serum  plus  the  sediment  from 
1  cc.  5%  guinea-pig  blood.  (Series  A.) 


622 


COLLECTED   STUDIES   IN  IMMUNITY. 
TABLE   II. 


Haemolysis  of  1  cc.  5%  Guinea-pig  Blood 
by  Inactivated  Ox  Serum  Plus  0.35  cc. 

Amount  of 

Horse  Serum. 

Ox  Serum. 

cc. 

A 

B 

Native  Horse 
Serum. 

Horse  Serum  Digested 
with  Guinea-pig 
Blood. 

0.25 

complete 

trace 

0.15 

'  ' 

faint  trace 

0.1 

(  t 

0 

0.05 

moderate 

0 

0.025 

trace 

0 

0 

0 

0 

In  a  control  series  (Table  II,  B),  the  sediment  from  1  cc.  5%  guinea-pig 
blood  was  mixed  with  0.35  cc.  horse  serum  plus  the  decanted  fluids  from  1  cc. 
5%  guinea-pig  blood.  The  latter  had  also  been  digested  with  decreasing 
amounts  of  inactive  ox  serum,  without,  however,  having  previously  been 
treated  with  horse  serum.  The  result  is  shown  in  Table  III. 


TABLE   III. 


Amount  of 
Inactive 
Ox  Serum, 
cc. 

Haemolysis  of  1  cc.  5%  Guinea-pig  Blood 
by  0.35  cc.  Horse  Serum  and 
Inactive  Ox  Serum. 

A 

Ox  Serum  Treated 
with  Blood  Pre- 
Digested  with 
Horse  Serum. 

B 

Ox  Serum  Treated 
with  Native  Blood. 

0.25 
0.15 
0.1 
0.05 
0.025 
0 

complete 

almost  complete 
moderate 
trace 
0 

complete 

almost  complete 
moderate 
trace 
0 

The  table  clearly  shows  that  the  guinea-pig  blood  does  not  absorb 
the  active  principle  of  the  ox  serum  even  when  the  blood  is  first 
digested  with  horse  serum.  We  were  able  to  confirm  the  result 
by  repeating  the  experiment  several  times.  The  assumption  of 
Bordet  and  Gay,  according  to  which  the  hypothetical  colloid  of 
ox  serum  (the  carrier  of  the  hsemolytic  action)  is  bound  by  blood- 
cells  which  have  been  digested  with  horse  serum,  is  thus  shown 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  623 

to  be  incorrect.  We  may  add  that  we  too  observed  marked  agglu- 
tination on  adding  ox  serum  to  the  guinea-pig  blood  previously 
treated  with  horse  serum.  Nevertheless,  on  testing  the  ox  serum 
separated  by  centrifuge,  we  found  that  this  still  possessed  all  its 
power  to  effect  haemolysis  of  guinea-pig  blood  in  conjunction  with 
horse  serum. 

By  this  we  do  not  intend  to  combat  the  statements  of  Bordet 
and  Gay,  that  guinea-pig  blood  treated  successively  with  horse 
serum  and  inactive  ox  serum  is  resistant  to  the  haemolytic  action 
of  the  active  mixture.  We  too  have  made  similar  observations, 
though  we  noted  that  haemolysis  was  absent  only  when  the  guinea- 
pig  blood-cells  had  been  treated  with  an  excess  of  horse  serum. 
Under  these  circumstances  it  was  immaterial  whether  the  pre- 
vious treatment  was  only  with  horse  serum  or  whether  treatment 
with  horse  serum  was  followed  by  digestion  with  inactive  ox  serum. 
It  is  to  be  noted,  however,  that  even  when  the  guinea-pig  blood- 
cells  were  found  resistant,  there  was  no  absorption  of  the  haemolytic 
component  of  the  ox  serum. 

In  the  following  experiment,  which  illustrates  the  conditions 
just  described,  we  first  determined  the  minimum  amounts  of  active 
horse  serum  and  inactive  ox  serum  which,  combined,  just  sufficed 
to  produce  complete  haemolysis.  This  dose  was  found  to  be  0.25 
cc.  for  each. 

Two  parallel  series  were  prepared.  To  1  cc.  5%  guinea-pig  blood  were 
added  decreasing  amounts  of  active  horse  serum.  The  mixtures  were  kept 
at  37°  for  one  hour,  and  then  centrifuged. 

Series  A.  The  sediments  of  series  A  were  digested  with  0.25  cc.  active  horse 
serum  and  02.5  cc.  inactive  ox  serum,  the  whole  being  made  up  to  about 
2.25  cc.  with  physiological  salt  solution. 

Series  B.  The  sediments  of  series  B  were  once  more  digested  for  one  hour 
at  37°  with  0.25  cc.  inactive  ox  serum  (and  salt  solution).  The  mixtures  were 
then  centrifuged  and  the  sediments  thus  obtained  mixed  with  0.25  cc.  horse 
serum  plus  0.25  cc.  inactive  ox  serum. 

Series  C.  The  supernatant  fluids  separated  by  centrifuge  in  series  B  were 
mixed  with  guinea-pig  blood  and  with  0.25  cc.  horse  serum.  (Total  volume 
about  2.25  cc.) 

The  result  is  shown  in  Table  IV. 

The  table  shows  that  the  guinea-pig  blood-cells  do  not  lose 
their  normal  susceptibility  when  they  are  treated  with  a  dose  of 
horse  serum  sufficient  to  produce  complete  haemolysis  (0.25  cc.). 


624 


COLLECTED   STUDIES  IN  IMMUNITY. 
TABLE   IV. 


Amount  of  Horse 
Serum  Used  for 

Haemolysis  of  1  cc.  5%  Guinea-pig 
Blood  in 

Blood-cells, 
cc. 

Series  A. 

Series  B. 

Series  C. 

1.0 
0.5 

trace 
moderate 

trace 
moderate 

complete 

0.25 
0.15 

complete 

(  e 

complete 

(  t 
f  i 

0.1 

tt 

<  t 

it 

0 

n 

1  1 

I  ( 

It  also  shows  that  an  excess  of  horse  serum  gives  rise  to  an  increased 
resistance  of  the  blood-cells  toward  what  is  otherwise  a  hsemolytic 
mixture,  and  this  effect  is  produced  whether  or  not  ox  serum  is 
subsequently  allowed  to  act  on  the  cells.  Moreover,  from  Column 
C,  we  see  that  in  this  case  also  the  ox  serum  has  not  lost  its  ability 
to  produce  haemolysis  in  conjunction  with  horse  serum.  Con- 
cerning the  cause  of  the  resistance  produced  by  treating  guinea- 
pig  blood-cells  with  horse  serum  alone  or  with  horse  serum  and 
ox  serum,  we  can  only  conjecture.  It  is  quite  possible  that  the 
effect  is  due  to  an  antagonism  between  agglutination  and  haemol- 
ysis, as  suggested  by  Bordet  and  Gay.  It  is  also  conceivable  that 
horse  amboceptor  and  ox  amboceptor  attack  the  same  receptors 
of  the  biood-cells,  and  that  preliminary  treatment  with  an  excess 
of  horse  serum  blocks  the  way  for  the  ox  amboceptor.  Be  this  as 
it  may,  our  experiments  show  that  the  ox  serum  component  con- 
cerned in  this  haemolysis  is  not  bound  when  the  guinea-pig  blood- 
cells  are  first  digested  with  active  horse  serum.  Bordet  and  Gay's 
assumption,  that  ox  serum  produces  this  haemolysis  through  a 
"  colloid "  constituent  which  acts  only  after  amboceptor  and  com- 
plement have  combined  with  the  guinea-pig  blood-cell,  must  there- 
fore be  abandoned.  On  the  other  hand,  Klein's  observation,  that 
guinea-pig  blood  previously  treated  with  horse  serum  is  no  longer 
dissolved  by  inactive  ox  serum,  is  readily  explained  in  accordance 
with  the  ideas  expressed  by  Browning.  The  horse  serum  comple- 
ment, though  not  dominant  for  the  horse  amboceptor,  is  anchored 
to  the  cell  by  means  of  this  amboceptor,  and  thus  is  no  longer 
available  for  the  ox  amboceptor.  We  have  tried  to  illustrate  the 
conditions  in  Figs.  1  and  2  of  the  accompanying  plate.  It  may  be 
added  that  in  this  case  it  is  impossible  to  produce  haemolysis  either 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  625 

by  employing  an  excess  of  ox  serum,  or  by  employing  the  min- 
imum complete  solvent  dose  of  horse  serum  for  the  preliminary 
treatment  of  the  guinea-pig  blood-cells.  In  contrast  to  this,  the 
blood-cells  which  have  been  previously  treated  with  horse  serum 
only  then  fail  to  hsemolyze  on  the  addition  of  ox  serum  plus  horse 
serum  when  the  amount  of  horse  serum  used  for  the  preliminary 
treatment  is  excessive.  As  we  have  seen,  Bordet  and  Gay  regarded 
the  resistance  of  the  cells  against  ox  serum  alone  and  against  the 
combined  action  of  ox  serum  and  horse  serum  as  having  a  common 
origin.  From  what  has  been  said  it  is  apparent,  however,  that 
these  phenomena  will  have  to  be  considered  separately.  In  the 
former  case  complement  is  absent,  and  the  inhibition,  is  therefore 
absolute.  In  the  latter  case  complement  is  present,  the  absence 
of  haemolysis  being  a  secondary  effect  dependent  on  quantitative 
relations.  In  the  case  described  by  Ehrlich  and  Sachs,  in  which 
guinea-pig  blood  is  haemolyzed  by  inactivated  ox  serum  and  horse 
serum,  we  do  not  see  the  least  reason  for  abandoning  the  explana- 
tion offered  by  the  authors.  According  to  this,  it  will  be  remem- 
bered, the  amboceptor  is  contained  in  the  ox  serum.  In  this  com- 
bination it  is  absolutely  unnecessary  to  assume  the  existence  of 
a  third  component  which  takes  part  in  the  ha?molytic  action. 

Nevertheless  we  sought  to  find  additional  evidence  to  show 
that  the  two  components  of  horse  serum  and  inactive  ox  serum 
were  directly  related  to  one  another,  or  that  the  amboceptor  con- 
tained in  horse  serum  played  no  part  in  the  haemolysis.  In  this 
we  were  successful  in  more  ways  than  one.  If  it  is  necessary  for 
ox  amboceptor  and  horse  complement  to  first  unite  and  form  an 
active  haemolysis  before  combining  with  the  cell  receptors,  we  should 
expect  that  haBmolysis  would  result  more  quickly  if  horse  serum 
and  ox  serums  were  digested  for  a  time  before  adding  the  blood, 
than  if  all  three  components  were  mixed  at  once.  We  therefore 
proceeded  as  follows: 

Two  series  of  tubes  were  prepared: 

Series  A.  Decreasing  amounts  of  horse  serum  (total  volume  0.75  cc.)  were 
kept  for  one  hour  at  37°.  Then  1  cc.  5%  guinea-pig  blood  plus  0.5  cc.  inactive 
ox  serum  are  added  to  each  tube. 

Series  B.  Decreasing  amounts  of  horse  serum  are  digested  for  one  hou? 
at  37°  with  0.5  cc.  inactive  ox  serum,  after  which  1  cc.  5%  guinea-pig  blood 
is  added  to  each  tube. 

The  degree  of  haemolysis  was  noted  at  the  end  of  5,  15,  and  30  minutes 
and  after  two  hours.  The  result  is  shown  in  Table  V. 


626 


COLLECTED  STUDIES  IN  IMMUNITY. 
TABLE   V. 


Degree  of  Haemolysis. 

Amount 

of 

Horse 

Series  A. 

Series  B. 

cc. 

5  Min. 

15  Min. 

30  Min. 

2Hrs. 

5  Min. 

15  Min. 

30  Min. 

2Hrs. 

0.75 

0 

0 

strong 

complete 

strong 

complete 

complete 

complete 

0.5 

0 

0 

moderate 

" 

" 

f    almost 
\  complete 

" 

" 

0.35 

0 

0 

slight 

stror^ 

0 

moderate 

/    almost 
\  complete 

" 

0.25 
0.15 

0 
0 

0 
0 

6 
0 

slight 
trace 

0 
0 

0 
0 

strong 
slight 

strong 
slight 

0.1 

0 

0 

0 

faint  trace 

0 

0 

0 

trace 

0.05 

0 

0 

0 

0 

0 

0 

0 

faint  trace 

0 

0 

0 

0 

0 

0 

0 

0 

0 

The  table  shows  that  haemolysis  is  actually  more  rapid  when 
horse  serum  and  inactive  ox  serum  are  first  allowed  to  remain  in 
contact  for  a  time.  During  this  time  the  ox  amboceptor  and  horse 
complement  have  entered  into  combination,  and  the  period  of 
incubation  preceding  haemolysis  is  thus  shortened.  Moreover, 
as  can  be  seen  from  the  table,  the  final  hamolytic  effect  may  also 
be  somewhat  greater  when  ambloceptor  and  complement  are  first 
digested  together.  The  reason  for  this  evidently  lies  in  a  slight 
impairment  of  the  horse  complement  as  a  result  of  the  one  hour's 
heating  to  37°,  the  combination  of  ox  amboceptor  and  horse  com- 
plement evidently  being  more  resistant.  It  need  hardly  be  men- 
tioned that  these  results  are  incompatible  with  the  colloid  theory. 

If  we  could  remove  the  amboceptors  of  horse  serum  it  would 
be  possible  to  demonstrate  directly  the  amboceptor  role  played 
by  the  ox  serum.  It  is  well  known  that  a  method  devised  by  Ehr- 
lich  and  Morgenroth1  enables  us  to  separate  the  amboceptor  and 
complement  of  an  active  serum.  Thus,  by  digesting  red  blood- 
cells  at  0°  with  an  active  serum,  it  will  be  found  that  only  ambo- 
ceptor has  been  bound;  the  complement  remains  in  the  fluid.  In 
the  case  of  the  normal  ha3molysins,  to  be  sure,  a  difficulty  arises 
from  the  fact  that  the  binding  of  amboceptor  at  0°  is  usually  in- 
complete, some  of  the  amboceptor  remaining  unbound.  So  in 
the  case  of  the  amboceptors  of  horse  serum,  we  know  from  the  work 
of  Browning  that  at  0°  guinea-pig  blood-cells  bind  them  only  up  to 


1  Ehrlich  and  Morgenroth,  Berliner  klin.  Wochenschr.,  1899.     See  also  this 
volume,  page  1. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  627 

a  certain  point.  The  portion  bound,  to  be  sure,  is  not  inconsider- 
able. It  is  to  be  noted,  however,  that  horse  serum  treated  with 
guinea-pig  blood  at  0°  loses  practically  none  of  its  power  to  effect 
haemolysis  in  conjunction  with  inactive  ox  serum.  According 
to  Bordet  and  Gay's  conception,  provided  that  any  considerable 
quantity  of  amboceptor  had  been  bound,  this  should  not  be  the 
case,  for  in  the  opinion  of  these  authors  the  horse  serum  plays  the 
role  of  amboceptor  in  the  haemolysis.  A  decrease  in  the  quantity 
of  amboceptor  should,  of  course,  manifest  itself  by  a  reduction 
in  the  haBmolytic  power.  It  might  be  objected  that  the  ambo- 
ceptor in  horse  serum  exists  in  excess,  and  that  therefore  it  was 
entirely  irrelevant  whether  a  portion  was  present  or  absent.  This 
objection,  however,  can  be  tested  experimentally.  Suppose,  for 
example,  that  the  horse  serum  digested  at  0°  with  guinea-pig  blood, 
still  contained  enough  amboceptor  to  produce,  in  conjunction 
with  inactive  ox  serum,  the  full  hsemolytic  effect  as  conceived  by 
Bordet  and  Gay.  It  is  obvious  that  when  such  a  serum  is  subse- 
quently treated  with  guinea-pig  blood  at  37°  the  impairment  in 
the  ability  to  bring  about  hsemolytic  effects  should  be  as  great  or 
even  greater  than  that  produced  in  native  horse  serum.  The 
experiment,  however,  shows  that  just  the  contrary  is  the  case.  The 
conditions  are  really  reflected  in  Table  2  of  Browning's  paper. 
We  shall,  however,  reproduce  the  result  of  an  analogous  experiment. 

Three  series  of  tubes  are  prepared,  each  containing  1  cc.  5%  guinea-pig 
blood  and  decreasing  amounts  of  horse  serum  diluted  with  the  same  amount 
of  physiological  salt  solution.  The  total  volume  in  each  tube  is  2  cc. 

The  tubes  of  series  A  are  kept  at  37°  for  1^  hours  and  then  centrifuged. 
The  supernatant  fluids  thus  obtained  are  then  digested  with  the  sediments 
from  1  cc.  5%  guinea-pig  blood  plus  0.3  cc.  inactive  ox  serum. 

The  tubes  of  series  B  are  centrifuged  after  having  been  kept  at  0°  for  two 
hours.  The  supernatant  fluids  are  treated  as  is  series  A. 

The  tubes  of  series  C  are  centrifuged  after  having  been  kept  at  0°  for  two 
hours.  The  supernatant  fluids  are  digested  for  two  hours  at  37°  with  the  sedi- 
ments from  1  cc.  5%  guinea-pig  blood.  After  again  centrifuging,  the  super- 
natant fluids  are  treated  with  the  sediments  from  1  cc.  5%  guinea-pig  blood 
plus  0.3  cc.  inactive  ox  serum. 

The  result  is  shown  in  Table  VI. 

The  horse  serum  which  underwent  a  preliminary  treatment 
at  0°  is  thus  seen  to  have  lost  but  little  of  its  power  to  bring  about 
haBmolysis,  by  the  subsequent  digestion  at  37°.  Certainly  the 
reduction  is  considerably  less  than  that  produced  by  the  direct 


628 


COLLECTED  STUDIES   IN  IMMUNITY. 


TABLE   VI. 


Haemolysis  of  1  cc.  5%  Guinea-pig  Blood  by  0.3  cc.  Inactive  Ox  Serum 

and  Horse  Serum. 

Amounts  of 
the  Half-diluted 
Horse  Serum. 

Previously  Treated  with  Guinea-pig  Blood 

Native. 

A. 

B. 

C. 

at  37°  C. 

at  0°  C. 

at  0°  +  37°. 

0.5 
0.25 

complete 

almost  complete 
strong 

complete 

complete 

<  < 

0.15 

(  i 

moderate 

it 

almost  complete 

0.1 

trace 

strong 

moderate 

0.05 

strong 

faint  trace 

moderate 

slight 

0.025 

moderate 

0 

slight 

trace 

0.0 

0 

0 

0 

0 

treatment  of  the  native  serum  at  37°.  This  is  all  the  more  notice- 
able because  in  the  above  table  a  slight  reduction  of  haemolytic 
power  is  shown  as  a  result  of  digestion  at  0°.  This  reduction  is 
probably  due  to  a  slight  loss  of  supernatant  fluid  in  decanting  the 
centrifugates.  The  result  of  the  experiment  is  absolutely  at  var- 
iance with  the  colloid  theory.  Assuming  that  the  horse  serum 
acts  both  as  amboceptor  and  complement,  while  the  ox  serum, 
in  accordance  with  the  view  of  Bordet  and  Gay,  furnishes  a  "col- 
loid "  which  takes  part  in  the  haemolysis,  it  follows  that  successive 
treatment  at  0°  and  37°  would  effect  a  greater  reduction  of  the 
active  principle  than  a  single  treatment  at  37°.  The  result,  on 
the  other  hand,  harmonizes  perfectly  with  the  view  expressed 
by  Ehrlich  and  Sachs,  and  could,  in  fact,  have  been  foretold  on  the 
basis  of  that  conception.  The  horse  serum  furnishes  only  the 
complement.  By  treatment  at  0°  a  portion  of  the  amboceptor  is 
removed,  so  that  the  serum  thus  becomes  rich  in  complement  but 
poor  in  amboceptor.  On  digesting  such  a  serum  once  more  with 
guinea-pig  blood,  at  37°,  a  small  amount  of  complement  is  removed 
through  the  intervention  of  what  amboceptor  still  remains.  The 
loss  of  complement  thus  sustained  is  bound  to  be  less  than  that 
observed  when  native  serum  (which  is  rich  in  amboceptor)  is  digested 
with  guinea-pig  blood.  Our  experimental  analysis  therefore 
shows  that  the  interpretation  offered  by  Bordet  and  Gay  cannot 
be  harmonized  with  the  facts.  In  fact  our  study  furnishes  addi- 
tional confirmation  for  the  view  that  in  the  case  under  discussion 
the  ox  serum  acts  as  an  amboceptor  with  the  horse  serum  as  com- 
plement. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  629 


III. 

Our  further  efforts  had,  naturally,  to  be  directed  to  a  study  of 
the  experiment  reported  by  Bordet  and  Gay  which  forms  so 
important  a  link  in  their  demonstration.  It  is  based  on  the  unique 
observation  that  ox  blood  laden  with  specific  amboceptor  does  not 
dissolve  in  horse  serum,  but  does  so  in  a  mixture  of  active  horse 
serum  and  inactive  ox  serum.  It  is  true  that  there  is  a  certain 
external  analogy  between  this  phenomenon  and  the  haemolysis  of 
guinea-pig  blood  by  the  same  mixture.  In  the  haemolysis  of  the 
sensitized  ox  blood  it  is  impossible  that  the  ox  serum  acts  as  ambo- 
ceptor, and  this  leads  Bordet  and  Gay  to  conclude  that  in  the 
haemolysis  of  the  guinea-pig  blood  the  ox  serum  does  not  act  as  an 
amboceptor.  We  have  already  seen  that  this  conclusion  is  not 
warranted.  It  was  felt,  however,  that  it  would  be  interesting  to 
inquire  more  closely  into  the  peculiar  mechanism  of  the  haemolytic 
action  in  the  ox-blood  combination,  the  more  so  since  the  view  of 
Bordet  and  Gay,  that  the  ox  serum  represents  a  "colloid"  which 
dissolves  the  blood-cells  previously  prepared  by  amboceptor  and 
complement,  is  an  assumption  devised  for  this  particular  case,  and 
one  which  would  constitute  an  entirely  new  kind  of  serum  haemolysis. 
We  therefore  sought  to  find  an  explanation  for  the  haemolysis  in 
in  question  on  the  basis  of  phenomena  previously  observed. 

In  our  experiments  we  used  an  inactivated  immune  serum 
derived  from  a  rabbit  which  had  been  immunized  with  ox  blood. 
One  cubic  centimeter  5%  ox  blood  was  just  completely  dissolved 
(in  the  presence  of  0.1  cc.  guinea-pig  complement)  by  0.0005  cc.  of 
this  specific  immune  serum.  In  order  to  effect  haemolysis  of  ox 
blood  by  the  mixture  "horse  serum  plus  inactive  ox  serum"  it  was 
necessary  to  use  0.05  cc.  amboceptor.  In  the  following  experiment, 
when  speaking  merely  of  prepared  ox  blood,  it  is  understood  that 
1  cc.  5%  ox  blood  was  treated  with  0.05  cc.  amboceptor.  Amounts 
smaller  than  this  did  not  suffice  for  complete  haemolysis,  and  larger 
amounts  had  to  be  avoided  because  then  even  small  amounts  of 
horse  serum  alone  would  produce  haemolysis.  In  fact  according 
to  our  experience  the  prepared  blood-cells  are  often  haemolyzed  to 
a  greater  or  less  extent  by  the  horse  serum  alone  when  this  is  used  in 
rather  large  doses.  This  frequently  makes  it  impossible  to  deter- 
mine the  close  of  horse  serum,  which  by  itself  is  inert  but  which  in 


630 


COLLECTED  STUDIES   IN   IMMUNITY. 


conjunction  with  inactive  ox  serum  still  produces  complete  haemolysis. 
Herein  we  see  the  first  difference  between  this  haemolysis  and  the 
haemolysis  of  guinea-pig  blood,  for  in  the  latter  the  horse  serum  was 
always  inert  or  only  feebly  haemolytic.  Moreover,  he  have  en- 
countered further  marked  differences  which  speak  strongly  against 
the  identity  of  the  mechanism  in  the  two  cases  which  Bordet  and 
Gay  cite  as  analogous.  Thus  it  was  found  that  an  excess  of  ox 
serum  inhibits  the  haemolysis  of  the  prepared  ox-blood  cells  by  horse 
serum  plus  ox  serum,  whereas  the  degree  of  haemolysis  of  the  guinea- 
pig  blood  cells  is  proportionate  to  the  amount  of  ox  serum.  This  is 
shown  in  the  following  experiment: 

Two  series  of  tubes  were  prepared: 

Series  A.  One  cc.  prepared  5%  ox  blood  plus  decreasing  amounts  of  inactive 
ox  serum  plus  0.15  cc.  horse  serum  (minimum  amount). 

Series  B.  One  cc.  5%  guinea-pig  blood  plus  decreasing  amounts  of  inactive 
ox  serum  plus  0.25  cc.  horse  serum  (minimum  amount). 

The  degree  of  haemolysis  is  shown  in  Table  VII. 


TABLE   VII. 


Amount  of  Inactive 
Ox  Serum. 

Series  A. 

Series  B. 

cc. 

1.0 

slight 

complete 

0.5 

moderate 

'  ' 

0.25 

almost  complete 

1  1 

0.1 

complete 

I  C 

0.05 

moderate 

moderate 

0.025 

slight 

slight 

0.01 

trace 

trace 

0 

0 

0 

The  behavior  of  the  ox  serum  in  the  two  series  is  totally 
different,  so  that  it  is  impossible  to  ascribe  the  action  of  the  serum  to 
one  and  the  same  cause.  According  to  Bordet  and  Gay,  however, 
the  ox  serum  in  both  cases  acts  neither  as  amboceptor  nor  as  com- 
plement, but  participates  in  the  reaction  as  a  colloid  as  already 
discussed.  From  this  standpoint  it  is  impossible  to  understand  the 
difference  in  the  behavior  or  the  ox  serum  in  the  two  series.  Looked 
at  from  our  point  of  view,  however,  the  difference  is  readily  explained, 
for  then  we  regard  the  ox  serum  as  acting  as  an  amboceptor  in  the 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  631 

haemolysis  of  guinea-pig  blood,  but  acting  in  quite  another  manner 
in  the  haemolysis  of  the  prepared  ox  blood. 

Another  difference  between  the  two  phenomena  is  presented 
by  the  following:  If  prepared  ox  blood-cells  are  successively  digested 
with  horse  serum  and  inactive  ox  serum,  no  haemolysis  occurs.  This 
is  entirely  analogous  to  what  is  observed  with  guinea-pig  blood- 
cells.  While,  however,  when  a^  large  amount  of  horse  serum  has 
been  used,  the  guinea-pig  blood-cells  are  resistant  to  the  combined 
action  of  horse  serum  and  inactive  ox  serum,  this  is  not  the  case 
with  the  prepared  ox  blood.  Before  going  into  details,  however, 
it  may  be  well  to  make  certain  general  observations  concerning  the 
behavior  of  the  components  in  the  haemolysis  of  prepared  ox  blood. 
Thus  it  was  found  that  to  be  impossible  to  replace  the  inactive  ox 
serum  by  hog  or  rabbit  serum.  The  same  was  true  for  inactive 
sheep  serum,1  whereas  inactive  goat  serum  in  conjunction  with  horse 
serum  acted  like  ox  serum  though  weaker.2  We  also  noted  the  effect 
of  thermic  influence  on  the  components  of  horse  serum  3  and  found 
that  the  ox  serum  could  be  heated  for  half  an  hour  to  55°  without 
affecting  its  action,  while  on  heating  for  half  an  hour  to  65°  it  lost 
its  power  to  dissolve  prepared  ox  blood  in  conjunction  with  horse 
serum.  So  far  as  the  relation  of  the  individual  components  to  the 
prepared  blood-cells  is  concerned,  it  was  found  that  active  horse 
serum  is  robbed  of  its  active  constituent  by  treatment  with  prepared 
blood.  In  fact,  not  only  does  it  thereby  lose  its  property  to  dissolve 
prepared  ox  blood  (confirming  Bordet  and  Gay),  but  it  also  ceases  to 
dissolve  guinea-pig  blood  in  conjunction  with  inactive  ox  serum 
(confirming  the  statements  of  Browning) .  This  was  to  be  expected, 
because  in  both  combinations  the  horse  serum  acts  as  complement, 
and  a  suitable  amboceptor  is  present.  In  both  cases,  therefore, 
the  amboceptor  can  effect  absorption  of  complement  without  giving 
rise  to  haemolysis.  There  is  another  point  of  agreement  between  the 
two  combinations.  Thus,  despite  the  anchoring  of  horse  com- 
plement brought  about  by  treatment  with  horse  serum,  the  prepared 
ox  blood-cells  do  not  dissolve  on  the  addition  of  inactive  ox  serum. 

1  Active  sheep  serum  by  itself  is  slightly  haemolytic  for  prepared  ox  blood. 
The  action  is  intensified,  however,  by  the  addition  of  horse  serum. 

2  It  should  be  remarked  that  in  the  haemolysis  of  guinea-pig  blood  the  ox 
serum  can  be  replaced  by  goat  serum.     The  mode  of  action  is  the  same  in 
both  cases. 

3  Ox  serum?— [Editor.] 


632 


COLLECTED   STUDIES   IN   IMMUNITY. 


Prepared  blood  so  treated,  however,  at  once  dissolves  in  a  mixture 
containing  minimum  quantities  of  horse  serum  and  inactive  ox 
serum.  This  is  illustrated  in  the  following  experiment: 

Two  similar  series  of  tubes  are  prepared.  The  tubes  in  each  series  contain 
1  cc.  5%  prepared  ox  blood  and  decreasing  amounts  of  active  horse  serum  (total 
volume  2  cc.)-  After  remaining  for  two  hours  at  37°  the  tubes  are  centrifuged. 
In  the  first  two,  containing  the  largest  amounts  of  horse  serum,  a  trace  of 
haemolysis  was  noticed. 

A.  The  supernatant  fluids  were  mixed  each  with  the  sediments  of  1  cc.  5% 
prepared  ox  blood,  plus  0,1  cc.  inactive  ox  serum.     (0.1  cc.  is  the  smallest  dose 
necessary  to  produce  complete  haemolysis.) 

B.  The  sediments  are  suspended  in  salt  solution  plus  0.1  cc.  inactive  ox 
serum. 

C.  The  sediments  are  suspended  in  salt  solution  plus  0.1  cc.  inactive  ox 
serum  plus  0.35  cc.  horse  serum. 

The  result  is  shown  in  the  following  table: 


TABLE  VIII. 


Amount  of 

Degree  of  Haemolysis  of  1  cc.  5%  Prepared  Ox  Blood  plus  0.1  cc.  Inactive 
Ox  Serum  plus  Horse  Serum. 

Horse  Serum. 

cc. 

Control. 

A. 

B. 

C. 

1.0 
0.5 

complete 

slight 
trace 

0 
0 

complete 

0.35 

1  1 

faint  trace 

0 

0.25 

strong 

0 

0 

0.15 

moderate 

0 

0 

0.1 

trace 

0 

0 

0 

0 

0 

0 

Column  B  of  the  table  shows  exactly  the  same  behavior  as  in  a 
corresponding  experiment  with  guinea-pig  blood.  Despite  the  fact 
that  horse  serum  has  bound  the  amboceptor  and  complement, 
there  is  no  haemolysis  on  the  addition  of  inactive  ox  serum.  One 
of  the  main  arguments  which  could  have  been  advanced  in  support  of 
Bordet-Gay's  colloid  theory  thus  fails.  It  is  also  apparent  that  no 
special  resistance  of  the  prepared  blood-cells  comes  into  question, 
for  in  column  C  we  find  that  these  cells  are  completely  dissolved  in  a 
suitable  mixture. 

Bordet  and  Gay,  to  be  sure,  do  say  that  prepared  ox  blood-cells 
treated  with  horse  serum  absorb  the  effective  principles  of  inactive 
horse  serum.  However,  all  that  they  describe  as  a  result  of  this  is  a 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS. 


633 


marked  agglutination;  they  say  nothing  about  the  occurrence  of 
haemolysis,  though  haemolysis  is  what  one  should  have  expected 
according  to  their  theory.  On  the  other  hand  the  authors  tell  us 
that  ox  serum,  by  acting  on  ox  blood  which  has  been  prepared  and 
loaded  with  horse  complement,  loses  its  power  to  agglutinate,  in 
conjunction  with  horse  serum,  prepared  ox  blood.  Nothing  is  said 
about  haemolytie  action.  According  to  the  authors  ox  serum  so 
treated  when  tested  in  conjunction  with  horse  serum  on  guinea-pig 
blood,  does  agglutinate  and  dissolve  the  blood  more  slowly  and 
more  feebly.  We  felt  it  advisable  to  study  the  conditions  more 
closely,  and  proceeded  along  the  lines  already  described  in  our 
analysis  of  guinea-pig  blood  haemolysis. 

Two  series  of  tubes  are  prepared.  Each  tube  contains  1  cc.  prepared  5% 
ox  blood  which  has  previously  been  treated  for  one  hour  with  0.5  cc.  horse 
serum  (  =  2  complete  haemolytic  doses)  at  37°  and  then  freed  from  fluid  by 
centrifuge.  Decreasing  amounts  of  inactive  ox  serum  are  added  to  each  tube, 
the  mixtures  kept  at  37°  for  one  hour  and  centrifuged.  The  decanted  fluids 
in  the  one  series  are  digested  each  with  the  sediments  from  1  cc.  5%  prepared 
ox  blood  plus  0.25  cc.  horse  serum,  and  in  the  other  series  with  the  sediments 
from  1  cc.  5%  guinea-pig  blood  plus  0.25  cc.  horse  serum.  The  result  is  shown 
in  the  following  table: 

TABLE  IX. 


Amount  of 
Inactive 
Horse  Serum. 

cc. 

Haemolysis  of 

1  cc.  5%  Prepared  Ox  Blood  by 
0.25  cc.  Horse  Serum  plus 

1  cc.  5%  Guinea-pig  Blood  by 
0.25  cc.  Horse  Serum  plus 

A. 

Treated  Ox 
Serum. 

B. 
Native  Ox 
Serum. 

A. 

Treated  Ox 
Serum. 

B. 
Native  Ox 
Serum. 

0.35 
0.25 
0.15 
0.1 
0.05 
0 

complete 

t  f 

strong 
moderate 
0 

complete 

«  < 

strong 
moderate 
0 

complete 
almost  complete 
strong 
slight 
faint  trace 
0 

complete 

almost  complete 

a 

moderate 
trace 
0 

From  the  table  it  can  be  seen  that  ox  blood  loaded  with  horse 
complement  is  likewise  unable  to  deprive  inactive  ox  serum  of  the 
constituent  which  brings  about  haemolysis.  In  fact  ox  blood  so 
treated  is  able,  in  conjunction  with  horse  serum,  to  dissolve  with 
full  or  only  slightly  impaired  power  not  only  prepared  ox  blood-cells 


634  COLLECTED   STUDIES  IN  IMMUNITY. 

but  also  those  of  the  guinea-pig.  In  this  respect,  therefore,  our 
results  are  somewhat  opposed  to  the  statements  of  Bordet  and  Gay. 
For  the1  sake  of  completeness  it  may  be  mentioned  that  ox  serum 
digested  with  guinea-pig  blood  which  has  previously  been  treated 
with  active  horse  serum  loses  nothing  of  its  power  to  bring  about 
haBmolysis  of  prepared  ox  blood. 

To  sum  up:  1.  Prepared  ox  blood  treated  with  active  horse 
serum  does  not  dissolve  in  inactive  ox  serum. 

2.  The  constituent  of  the  ox  serum  which  brings  about  haemolysis 
is  not  absorbed  by  prepared  ox  blood  previously  treated  with  horse 
serum. 

This  shows  that  the  haemolysis  of  prepared  ox  blood  by  the  com- 
bined action  of  inactive  ox  serum  and  active  horse  serum,  as  also 
the  haemolysis  of  guinea-pig  blood  under  the  same  conditions  cannot 
be  explained  on  the  basis  of  the  colloid  theory  of  Bordet  and  Gay. 
We  have  seen  that  the  simplest  postulates  of  this  theory  cannot  be 
verified  experimentally.  In  the  ha3molysis  of  guinea-pig  blood  it  is 
at  once  clear  that  it  is  not  the  horse  serum,  as  Bordet  and  Gay 
suppose,  but  the  ox  serum  which  furnishes  the  haemolytic  amboceptor. 
This  ox  amboceptor,  as  Ehrlich  and  Sachs  have  shown,  is  peculiar 
in  that  it  requires  first  to  be  united  with  horse  complement  before 
it  can  be  anchored  by  the  red  blood-cells. 

In  explaining  the  haemolysis  of  prepared  ox  blood,  it  is  impossible 
to  regard  the  ox  serum  as  acting  as  an  amboceptor,  and  Bordet  and 
Gay  have  very  properly  called  attention  to  this  fact.  One  might 
perhaps  think  that  the  inactivated  ox  serum  acts  as  a  complementoid. 
In  that  case,  to  be  sure,  the  function  of  the  complementoid  would  be 
rather  peculiar.  It  would  be  necessary  to  assume  that  the  active 
horse  complement  was  bound  by  the  amboceptor-laden  blood-cells 
at  an  unsuitable  point  so  that  the  complement  could  not  exert  its 
action,  or,  in  other  words,  so  that  it  was  "not  dominant."  The 
role  of  the  ox  complementoid  would  then  consist  in  directing,  as  it 
were,  the  horse  complement  in  the  right  direction.  One  could,  for 
instance,  imagine  that  the  complementoid  possessed  a  higher  affinity 
than  the  horse  complement,  and  that  it  would  thus  block  the  ambo- 
ceptor group  at  which  the  complement  is  not  dominant.  The  horse 
complement  would  thus  be  anchored  by  the  complementophile 
amboceptor  group  for  which  it  really  possesses  the  smaller  affinity 
but  at  which  it  is  dominant.  Still  other  interpretations  are  possible, 
but  it  would  always  be  necessary  to  assume  that  the  ox  complementoid 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS. 


635 


is  already  itself  bound  by  the  prepared  ox  blood.  It  can,  however, 
be  shown  that  the  active  principle  of  the  ox  serum  loses  none  of  its 
power  by  digestion  with  prepared  ox  blood-cells.  From  this  it 
follows  that  the  view  just  discussed,  wherein  the  ox  serum  is  regarded 
as  acting  as  a  complementoid,  is  incorrect. 

It  was  necessary  to  cast  about  for  other  explanations,  and  it  was 
natural  to  think  that  in  the  haemolysis  of  the  prepared  ox  blood  too, 
the  inactive  ox  serum  possessed  direct  relations  to  the  horse  serum. 
We  had  noticed  that  the  ox  serum  amboceptor  acting  on  guinea-pig 
blood  possessed  a  marked  affinity  for  horse  complement.  This  fact 
suggested  that  the  ox  serum  could  produce  anticomplementary 
effects,  for  it  is  readily  understood  that  an  amboceptor  possessing 
affinity  for  the  complement  will  act  like  an  anticomplement  when  the 
suitable  blood-cells  are  absent.  As  a  matter  of  fact  we  have  shown 
(see  Table  VII)  that  large  amounts  of  inactive  ox  serum  hinder  the 
ha3molysis  of  the  prepared  ox  blood.  This  inhibition  can  only  be 
due  to  anticomplement  action.  These  findings  naturally  led  us  to 
suspect  that  the  inactive  ox  serum  and  the  horse  serum  were  in 
some  way  related  to  one  another  in  the  production  of  the  hsemolytic 
effect.  We  therefore  proceeded  as  follows: 

In  one  series  of  tubes  decreasing  amounts  of  horse  serum  were  kept  for 
one  hour  at  37°,  whereupon  prepared  ox  blood  plus  0.5  cc.  inactive  ox  serum 
were  added.  In  another  series  decreasing  amounts  of  horse  serum  were  digested 
for  one  hour  with  0.5  cc.  inactive  ox  serum  at  37°  whereupon  the  blood-cells 
were  added.  The  degree  of  haemolysis  was  noted  from  time  to  time,  and  is 
is  shown  in  the  following  table: 


TABLE  X. 


Haemolysis  of  1  cc.  5%  Prepared  Ox  Blood  by  Horse  Serum  plus  Inactive 

Ox  Serum. 

Amounts 

Active 

Horse 
Serum, 
ec. 

A.  Horse  Serum  Alone  1  Hour  at  37°. 

B.  Horse  Serum  plus  Ox  Serum 
1  Hour  at  37°. 

5  Min. 

15  Min. 

30  Min. 

2  Hours. 

5  Min. 

15  Min. 

30  Min. 

2  Hours. 

0.75 

0 

complete 

complete 

complete 

complete 

complete 

complete 

complete 

0.5 

0 

moderate 

f    almost 
\  complete 

•• 

•« 

•• 

«« 

" 

0.35 

0 

slight 

moderate 

f    almost 
\complete 

strong 

" 

•« 

«• 

0.25 

0 

0 

0 

strong 

moderate 

moderate 

strong 

strong 

0.15 
0.1 

0 
0 

0 
0 

0 
0 

moderate 
trace 

0 
0 

slight 

slight 

moderate 
trace 

0.05 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

636  COLLECTED   STUDIES   IN  IMMUNITY. 

The  table  shows  the  same  condition  which  we  have  already  noted 
in  the  haemolysis  of  guinea-pig  blood.  The  haemolysis  of  the  prepared 
ox  blood  too,  proceeds  more  rapidly  if  the  horse  serum  and  inactive 
ox  serum  are  mixed  some  time  before  the  addition  of  the  blood-cells. 
From  this  it  follows  that  some  sort  of  a  reaction  takes  place  between 
constituents  of  the  horse  serum  and  of  the  ox  serum.  A  really 
active  complex  as  in  the  haemolysis  of  guinea-pig  blood  cannot  thus 
be  formed,  for,  as  we  have  repeatedly  pointed  out,  the  ox  serum 
cannot  functionate  as  an  amboceptor.  We  shall  probably  not  err 
if  we  assume  that  the  inactive  ox  serum  participates  in  the  haemolysis 
of  the  prepared  ox  blood  by  anchoring  a  constituent  of  horse  serum 
which  inhibits  the  action  of  the  horse  complement  responsible  for 
haemolysis.  An  autoanticomplement  of  horse  serum  is  out  of  the 
question,  if  only  for  the  reason  that  the  horse  complement  is  bound 
by  the  prepared  ox  blood-cells.  On  the  other  hand  it  seemed  very 
possible  that  the  horse  serum  constituent  in  question  which  inhibits 
haemolysis  and  which  is  bound  by  ox  serum,  possessed  the  character 
of  a  complement  or  a  complementoid.  The  action  of  this  second 
complement  of  horse  serum  would  be  this,  that  it  does  not  dissolve 
prepared  ox  blood,  but  possesses  a  higher  affinity  than  the  effective 
complement.  The  anchoring  of  this  constituent  would  cause  the 
effective  complement  to  be  bound  at  an  unsuitable  situation  where 
it  is  not  dominant.  In  order  to  prove  the  correctness  of  this  view 
it  is  necessary  to  show  that  the  binding  of  the  effective  horse  com- 
plement to  the  prepared  ox  blood,  and  the  haemolysis  of  prepared  ox 
blood  by  the  joint  action  of  active  horse  serum  and  inactive  ox 
serum,  are  two  independent  reactions.  In  other  words  we  must 
effect  a  binding  of  the  active  principle  of  horse  serum  and  yet  have 
no  haemolysis  when  under  exactly  the  same  conditions  inactive  ox 
serum  is  also  present.  This  we  have  succeeded  in  doing.  It  is 
very  easy  to  fulfil  the  conditions  just  mentioned,  by  digesting  the 
ox  blood  with  a  smaller  quantity  of  amboceptor.  We  proceeded  as 
follows : 

Two  series  of  tubes  are  prepared,  each  tube  containing  1  cc.  5%  ox  blood 
and  decreasing  amounts  of  amboceptor  (inactivated  serum  of  a  rabbit  immunized 
against  ox  blood).  After  remaining  at  37°  for  one  hour,  the  mixtures  were 
centrifuged.  The  sediments  were  then  treated  as  follows: 

Series  A.  Digested  with  a  mixture  of  0.2  cc.  horse  serum  plus  0.1  cc.  inactive 
ox  serum. 

Series  B.  Digested  with  0.2  cc.  horse  serum  l  for  one  hour  at  37°,  centrifuged, 
and  the  sediments  thus  obtained  mixed  each  with  0.1  cc.  inactive  ox  serum. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS. 


637 


/Series  C.  The  supernatant  fluids  separated  in  B  are  digested  with  the 
sediments  each  of  1  cc.  5%  prepared  ox  blood  (prepared  in  the  usual  way  with 
0.05  cc.  amboceptor)  plus  0.1  cc.  inactive  ox  serum. 

The  result  is  shown  in  the  following  table: 


TABLE   XI. 


Degree  of  Haemolysis. 

Amount  of  Amboceptor 
Used  for  the 
Preliminary 
Treatment. 

cc. 

Series  A. 
More  or  Less  Highly 
Prepared  Ox  Blood  + 
0.2  cc.  Horse  Serum 
+  0.1  cc.  Inactive 
Ox  Serum. 

Series  B. 
More  or  Less  Highly 
Prepared  Ox  Blood  + 
0.2  cc.  Horse  Serum 
Centrifuged,  +0.1  cc. 
Inactive  Ox  Serum. 

Series  C. 
Highly  Prepared 
Ox  Blood  (0.05)   + 
0.1  cc.  Inactive 
Ox  Serum  +  0.2  cc. 
Previously  Digested 
Horse  Serum 

0.1 

complete 

0 

faint  trace 

0.05 

'• 

0 

0.025 

strong 

0 

0.015 

slight 

0 

0.01 

trace 

0 

0.005 

0 

0 

0.0025 

0 

0 

slight 

0 

0 

0 

complete 

Total  volume  always  2  cc. 

The  table  shows  that  so  far  as  the  binding  of  horse  complement 
is  concerned,  ox  serum  which  has  been  prepared  with  one-tenth  the 
amount  of  amboceptor  (0.005  cc.)  behaves  exactly  the  same  as 
that  which  has  been  highly  prepared  (0.05  cc.  amboceptor).  In 
spite  of  this,  we  see  that  such  feebly  prepared  ox  blood  is  resistant  to 
the  combined  action  of  horse  serum  and  inactive  ox  serum  (Series  A) . 
Furthermore,  from  Series  B  it  is  apparent  that  the  successive  addi- 
tion of  horse  serum  and  inactive  ox  serum  does  not  lead  to  haemo- 
lysis. The  conditions  discussed  above  have  thus  been  fulfilled, 
and  the  result  shows  that  the  phenomenon  of  the  binding  of  horse 
complement  must  be  considered  apart  from  that  of  its  haemolytic 
action. 

The  following  is  probably  the  simplest  conception  we  can  make 
of  the  mechanism  of  the  entire  phenomenon.  In  view  of  the  multi- 
plicity of  amboceptors  in  a  given  immune  serum  (see  especially  the 
studies  of  Ehrlich  and  Morgenroth)  there  is  no  reason  why  we  should 
not  be  dealing  with  two  different  fractions  of  amboceptor  in  the 
immune  serum  used  to  prepare  the  ox  blood.  One  amboceptor  is 
present  in  high  concentration  and  binds  the  horse  complement, 
although  the  complement  is  not  dominant  for  this  amboceptor. 


638  COLLECTED  STUDIES  IN   IMMUNITY. 

The  other  amboceptor  is  present  in  much  smaller  amount,  and  is  the 
amboceptor  for  which  the  horse  complement  is  dominant.  This 
explains  how  a  small  amount  of  amboceptor  binds  complement,  and 
how  haemolysis  is  effected  only  with  a  considerable  excess  of  immune 
serum.  The  relations  existing  between  weak  and  strong  concen- 
tration of  amboceptor  in  the  immune  serum  are  to  a  certain  extent 
analogous  to  those  existing  between  horse  and  ox  amboceptor  in  the 
haemolysis  of  guinea-pig  blood.  There  is,  however,  an  important 
difference.  In  the  haemolysis  of  guinea-pig  blood  the  affinity  of  the 
ox  amboceptor  to  the  horse  complement  exceeds  that  of  the  horse 
amboceptor.  When  both  amboceptors  are  present,  therefore, 
haBmofysis  occurs.  In  the  haBmolysis  of  the  prepared  ox  blood, 
however,  it  is  not  sufficient  that  both  amboceptors  are  present,  for 
under  these  circumstances,  apparently,  the  complement  is  still 
anchored  by  the  amboceptor  for  which  it  is  not  dominant.  In  order 
that  the  complement  may  lay  hold  of  the  other  amboceptor,  the 
cooperation  of  the  inactive  ox  serum  is  necessary.  This  serum,  as  we 
have  seen,  must  have  direct  relations  with  the  horse  serum.  The 
only  way  in  which  we  can  conceive  of  this  relation  is  to  assume 
that  the  ox  serum  binds  a  horse  serum  constituent  of  complement 
character  which  directs  the  effective  horse  complement  toward  the 
amboceptor  unsuited  for  producing  haBmolysis.  The  principle 
underlying  this  explanation  is  not  new,  similar  relations  having  been 
studied  by  Ehrlich  and  Marshall.1  In  a  combination  described  by 
these  authors,  it  was  shown  that  the  union  of  a  certain  non-dominant 
complement  did  not  occur  until  after  another  complementophile 
group  of  the  amboceptor  had  bound  the  particular  complement 
which  was  dominant  in  this  case.  It  is  possible  that  we  are  here 
dealing  with  an  analogous  phenomenon. 

If  we  succeed,  therefore,  in  removing  the  constituent  of  horse 
serum  which  causes  the  effective  horse  complement  to  combine  with 
the  unsuited  amboceptor  (and  this,  as  we  have  seen,  is  accom- 
plished by  the  action  of  ox  serum),  we  permit  the  horse  complement 
to  unite  with  the  other,  effective,  amboceptor  and  hamolysis  can 
occur.  In  this  case,  however,  it  follows  that  the  binding  of  the  horse 
complement  to  the  weakly  prepared  ox  blood  will  not  occur  if  the 
horse  serum  constituent  which  brings  about  this  binding  is  rendered 


1  Ehrlich  and  Marshall,   Berliner  klin.   Wochenschrift,   No.   25,    1902.     See 
also  this  volume,  page  226. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS 


639 


inert  by  the  ox  serum.  This  we  were  actually  able  to  prove  ex- 
perimentally. Constituting  as  it  does  the  crucial  experiment  for 
testing  the  correctness  of  the  views  here  developed,  the  following, 
experiment  deserves  the  closest  attention. 

Two  series  of  tubes  are  prepared: 

Series  A.  Each  tube  contains  0.35  cc.  horse  serum  made  up  to  1.1  cc.  with 
salt  solution.  The  mixtures  are  kept  at  37°  for  half  an  hour,  and  then  digested 
for  1^  hours  at  37°,  each  with  the  sediments  from  1  cc.  5%  weakly  prepared 
(0.005  cc.  aniboceptor)  ox  blood.  Then  centrifuge.  The  decanted  fluids  are 
mixed  with  decreasing  amounts  of  inactive  ox  serum  (1  cc.  volume)  and  these 
mixtures  are  poured  each  over  the  sediments  from  1  cc.  5%  strongly  prepared 
(0.05  cc.  amboceptor)  ox  blood. 

Series  B.  Each  tube  contains  0.35  cc.  horse  serum  plus  decreasing  amounts 
of  inactive  ox  serum  (total  volume  1.1  cc.).  After  remaining  at  37°  for  half 
an  hour  the  mixtures  are  digested  for  1£  hours  at  37°,  each  with  the  sediments 
from  1  cc.  5%  weakly  prepared  (0.005  cc.  amboceptor)  ox  blood.  After 
centrifuging,  the  decanted  fluids  are  poured  each  over  the  sediments  from 
1  cc.  strongly  prepared  (0.05  cc.  amboceptor)  5%  ox  blood  and  1  cc.  salt 
solution  is  added. 

The  result  is  shown  in  the  following  table: 


TABLE   XII. 


Haemolysis  of  1  cc.  5%  Strongly 

Prepared  Ox  Blood. 

Amount  of 
Inactive 
Ox  Serum, 
cc. 

Series  A. 
By  Ox  Serum,  and 
Horse  Serum 
which  has  been 
Treated  with  Weakly 
Prepared  Blood. 

Series  B. 
By  Mixtures  of  Ox 
Serum  and  Horse 
Serum  after  the 
Mixtures  had  been 
Treated  with  Weakly 
Prepared  Blood. 

0.75 

trace 

complete 

0.5 

(I 

0.35 

t  ( 

0.25 

strong 

0.15 

moderate 

0.1 

trace 

0 

0 

0 

An  examination  of  the  table  makes  it  clear  that  the  horse  com- 
plement is  not  bound  to  the  weakly  prepared  ox  blood  when  sufficient 
quantities  of  the  inactive  ox  serum  are  added  to  the  horse  serum. 
This  result  shows  at  once  how  entirely  untenable  is  the  theory  of 
Bordet  and  Gay.  According  to  their  view  we  would  have  every 


640  COLLECTED  STUDIES  IN  IMMUNITY. 

reason  to  expect  haemolysis  in  Series  B  to  be  weaker  than  in  Series 
A.  Under  no  circumstances  could  it  be  stronger.  In  Series  B 
conditions  are  such  that  the  "  colloid "  of  these  authors  would  have 
every  opportunity  to  be  absorbed  by  the  weakly  prepared  blood 
laden  with  complement.  The  result,  however,  is  exactly  the  reverse, 
and  absolutely  contradicts  the  colloid  theory.  On  the  other  hand  the 
result  it  what  was  to  be  expected  in  accordance  with  our  view. 
The  table  clearly  shows  that  the  ox  serum  hinders  the  binding  of  the 
horse  complement  by  the  weakly  prepared  ox  blood.  Proceeding 
from  this  fact  we  arrive  at  an  understanding  of  the  part  played  by 
the  ox  serum  in  the  haemolysis  of  stongly  prepared  ox  blood  by  horse 
serum.  We  are  dealing  with  rather  complicated  relations  and  we 
have  therefore  thought  it  wise  to  represent  these  in  the  attached 
diagram,  figures  3-7. 

Fig.  3  represents  the  constitution  of  the  immune  serum.  Ambo- 
ceptor  a  is  present  in  weak  concentration,  while  the  other,  ambo- 
ceptor  6,  is  present  in  strong  concentration. 

Fig.  4.  pictures  our  conception  of  the  relations  existing  when 
strongly  prepared  ox  blood-cells  are  digested  with  horse  serum. 
The  immune  serum  used  for  preparing  the  blood  contains  two 
types  of  amboceptor,  namely  the  strongly  concentrated  amboceptor 
6,  and  the  weakly  concentrated  amboceptor  a.  (See  Fig.  3.)  The 
latter  is  the  amboceptor  for  which  the  horse  complement  ca,  is 
dominant.  The  horse  serum,  however,  contains  another  substance 
having  complementary  properties,  c/?  and  this  possesses  marked 
affinity  for  the  complementophile  group  J3  of  amboceptor  b.  Ambo- 
ceptor b  also  possesses  a  group  a  which  ordinarily  does  not  react 
with  ca.  Through  the  anchoring  of  component  cfi  to  /?  the  affinity 
of  this  group  ca  of  amboceptor  6  is  increased  so  that  now  ambo- 
ceptor 6  lays  hold  on  the  effective  complement  ca  with  great  avidity. 
Since,  however,  complement  ca  is  not  dominant  for  amboceptor  b, 
no  haemolysis  ensues. 

Fig.  5  illustrates  the  action  of  the  ox  serum  constituent  r.  This 
binds  c/?,  whereby  the  increased  affinity  of  group  a  of  amboceptor  b 
fails  to  occur.  This  in  turn  causes  ca  to  unite  with  a  thus  giving 
rise  to  haemolysis. 

If  amboceptor  a  is  absent,  i.e.,  if  the  ox  blood  has  been  weakly 
prepared,  it  will  be  understood  that  in  the  digestion  with  horse  serum, 
amboceptor  b  binds  c/?  and  through  this  also  ca.  The  decanted  fluid 
is  therefore  unable  to  dissolve  strongly  prepared  blood  even  when 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  641 

inactiveox  serum  is  present.  (See  Fig.  6  and  also  Table  12,  A,  of 
the  text.) 

Furthermore,  if  the  weakly  prepared  blood,  which  then  has  only 
bound  amboceptor  b,  is  digested  with  the  mixture  of  horse  serum 
and  inactive  ox  serum,  no  haBmolysis  occurs  because  the  effective 
amboeeptor  a  is  absent.  Since,  however,  the  ox  component  r  binds 
c/?,  ca  is  left  free.  In  this  case  if  the  decanted  fluid  is  poured  over 
strongly  prepared  ox  blood,  it  will  be  found  that  hsmolysis  occurs 
without  any  further  addition.  (See  Fig.  7,  and  experiment  Table 
12,  B.) 

Naturally,  in  addition  to  the  factors  described  above,  the  effects 
of  mass  action  must  be  considered.  Thus  if  a  small  quantity  of  ox 
serum  is  made  to  react  with  a  great  excess  of  amboceptor  6,  it  is 
evident  that  the  reaction  between  b  and  c  can  still  take  place.  It  will, 
however,  be  slower  and  less  complete  than  when  the  ox  serum  is 
entirely  absent.  If  then  amboceptor  a  is  present  at  the  same  time 
it  will  be  understood  that  a  portion  of  c  will  still  find  opportunity  to 
combine  with  it  so  that  haBmoylsis  occurs.  But  when  amboceptor  a 
is  absent,  that  is  when  the  ox  blood  is  weakly  prepared,  c  will  still 
be  able  to  combine  with  amboceptor  b  and  the  decanted  fluid  will 
have  lost  its  hsemolytic  power.  This  explains  a  point  in  Table  12. 
In  the  control  which  consisted  of  simple  mixtures  of  strongly  pre- 
pared ox  blood,  0.35  cc.  horse  serum,  and  decreasing  amounts  of 
inactive  ox  serum,  it  was  found  that  0.1  cc.  of  the  inactive  ox  serum 
still  produced  complete  haemolysis.  In  Table  12,  B,  on  the  other 
hand,  weakly  prepared  ox  blood  deprived  a  mixture  of  0.1  cc.  inactive 
ox  serum  plus  0.35  cc.  horse  serum  of  its  hsemolytic  power. 

Contrariwise  we  should  expect  to  find  the  effective  horse  com- 
plement kept  intact  after  digestion  with  weakly  prepared  ox  blood 
provided  the  excess  of  inactive  ox  serum  is  allowed  to  act  at  the  same 
time.  This  is  well  shown  in  the  following  experiment: 

Two  series  of  tubes  are  prepared: 

Series  A.  Each  tube  contains  0.5  cc.  weakly  prepared  10%  ox  blood  plus 
0.5  cc.  salt  solution  plus  decreasing  amounts  of  active  horse  serum.1 

Series  B.  Each  tube  contains  0.5  cc.  weakly  prepared  10%  ox  blood  plus 
0.5  cc.  inactive  ox  serum  plus  decreasing  amounts  active  horse  serum.2 

The  mixtures  are  kept  for  H  hours  at  37°  and  then  centrifuged.  The  slight 
amount  of  haemolysis  observable  in  series  B  is  shown  in  Table  XIII. 

1  Horse  serum  plus  salt  solution  previously  kept  at  37°  for  one  hour. 

2  Horse  serum  plus  ox  serum  previously  kept  at  37°  for  one  hour. 


642 


COLLECTED  STUDIES  IN   IMMUNITY. 
TABLE   XIII. 


Amount  of 
Horse  Serum 
cc. 

Haemolysis  of  1  cc.  5%  Weakly  Prepared 
Ox  Blood  by  Decreasing  Amounts 
of  Horse  Serum. 

A. 

By  Itself. 

B. 

Together  with  0.5 
cc.  Inactive  Ox 
Serum. 

0.75 
0.5 
0.35 
0.25 
0.15 
0.1 
0 

0 
0 
0 
0 
0 
0 
0 

strong 
slight 
0 
0 
0 
0 
0 

After  this  the  fluid  decanted  from  the  tubes  of  series  A  are  mixed  each 
with  0.5  cc.  inactive  ox  serum,  and  the  fluids  from  series  B,  each  with  0.5  cc. 
salt  solution.  The  mixtures  are  then  digested  each  with  the  sediments  from 
1  cc.  5%  strongly  prepared  ox  blood. 

In  control  series  C  made  at  the  same  time,  mixtures  containing  each  0.5  cc. 
inactive  ox  serum  plus  decreasing  amounts  of  horse  serum  were  digested  at 
37°  for  two  hours,  after  which  strongly  prepared  ox  blood  was  added. 

The  result  of  the  experiment  is  shown  in  the  following  table: 


TABLE   XIV. 


Amount  of 


Haemolysis  of  Ice.  5%  Strongly  Prepared  Ox  Blood. 


cc. 

Series  A. 

Series  B. 

Series.  C. 

0.75 

strong 

complete 

complete 

0.5 

slight 

*  ' 

0.35 

trace 

" 

1  1 

0.25 
0.15 

0 
0 

almost  complete 
strong 

almost  complete 
slight 

0.1 

0 

moderate 

trace 

0 

0 

0 

0 

From  the  table  it  is  clearly  apparent  that  in  the  digestion  with 
weakly  prepared  ox  blood,  the  horse  complement  remains  entirely 
intact  provided  plenty  of  ox  serum  is  present,  whereas  by  itself  it 
is  bound  by  the  prepared  blood,  as  can  be  seen  from  Column  A. 
The  evidence  presented  by  this  marked  difference  becomes  still 
stronger  through  the  fact  that  the  action  of  mixtures  of  horse  serum 
and  ox  serum  on  weakly  prepared  blood  results  in  a  slight  degree  of 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  643 

haemolysis  (See  Table  13,  B).  Despite  the  occurrence  of  this 
haemolysis  in  which  at  least  some  material  has  been  used  up,  the 
final  result  is  just  the  opposite  of  what  was,  a  priori,  to  have  been 
expected.  This  furnished  a  weighty  argument  in  favor  of  the  view 
we  have  brought  forward.  We  shall  probably  not  err  if  we  assume 
that  the  horse  serum  constituent  eft  is  a  complement,  but  that  it  is 
dominant  neither  for  amboceptor  a  nor  amboceptor  b.  The  ox  serum 
thus  plays  merely  the  part  of  anticomplement.  The  amboceptors 
of  ox  serum  in  general  evidently  possess  a  high  affinity  in  their 
complementophile  groups.  It  will  be  recalled  that  we  have  actually 
demonstrated  this  in  the  case  of  the  amboceptor  acting  on  guinea- 
pig  blood  and  complemented  by  horse  serum.  A  little  consideration, 
however,  will  show  that  such  amboceptors,  when  the  cells  on  which 
they  act  are  missing,  will  exert  an  anticomplementary  action.  This 
also  explains  how  the  inactivated  ox  serum  when  in  excess,  can 
inhibit  the  haemolysis  of  strongly  prepared  ox  blood  by  horse  com- 
plement ca,  although  this  same  ox  serum,  in  smaller  quantities, 
brings  this  haemolysis  about.  This  observation  has  been  repeatedly 
made  by  us.  It  is  merely  necessary  to  assume  that  ox  serum  also 
contains  very  small  quantities  of  complementophile  groups  a. 
Large  doses  of  the  serum  would  then  also  exert  a  deflecting  influence 
on  complement  ca. 

So  far  as  the  two  complements  of  horse  serum  are  concerned 
(cce  and  cfi)  it  seems  as  though  their  quantitative  relations  are 
subject  to  certain  fluctuations.  We  have  already  called  attention 
to  the  fact  that  horse  serum  alone  dissolves  prepared  ox  blood  cells 
to  a  van-ing  degree.  In  the  light  of  what  has  been  said  it  is  obvious 
that  the  haemolysis  produced  by  horse  serum  alone  will  be  stronger 
the  more  the  concentration  of  the  horse  complement  ca  exceeds 
that  of  complement  cp.  If  complement  c/?  were  entirely  absent  we 
should  find  that  the  haemolysis  produced  by  horse  serum  alone  would 
be  as  strong  as  that  produced  by  the  combined  action  of  horse  serum 
and  inactive  ox  serum.  We  have  not  met  with  such  extreme  cases. 
Nevertheless  we  have  observed  horse  sera  which  by  themselves 
produced  complete  haemolysis  of  prepared  ox  blood  in  doses  of  0.35 
to  0.3  cc.  while  the  addition  of  inactive  ox  serum  reinforced  com- 
plete haemolysis  only  to  the  extent  of  a  dose  of  0.15  cc.  horse  serum. 
We  see,  therefore,  that  a  critical  study  of  the  experimental  findings 
leads  to  conclusions  which  fit  perfectly  into  the  interpretation  we 
have  elaborated. 


644  COLLECTED  STUDIES   IN   IMMUNITY. 


EXPLANATION   OF   THE   FIGURES   ON  THE   PLATE. 

FlQS.  1  and  2  illustrate  the  haemolysis  of  guinea-pig  blood  by  the  combined 

action  of  active  horse  serum  and  inactive  ox  serum. 
2  =  guinea-pig  blood-cell;    ar  =  amboceptor  of  ox  serum;    ap  =  amboceptor  of 

horse  serum;  c  =  complement  of  horse  serum. 

FIG.  1  represents  the  conditions  obtaining  when  blood,  horse  serum,  and  ox 
serum    are    mixed    simultaneously.      The    ox    amboceptor  (ar)    combines 
with  the  horse  complement  (c)  and  thus  brings  about  haemolysis. 
FIG.  2. — The  guinea-pig  blood  is  first  digested  with  horse  serum  (ap  +  c).     The 
blood-cell  absorbs  the  horse  amboceptor  (ap)  and  this  in  turn  anchors  horse 
complement  (c).     The  ox  amboceptor  (ar)  subsequently  added  does  not  find 
any  horse  complement  (c)  at  its  disposal,  and  haemolysis  therefore  does  not 
occur. 
FIGS.  3-7  illustrate  the  haemolysis  of  ox  blood  laden  with  amboceptor,  by  the 

combined  action  of  active  horse  serum  and  inactive  ox  serum. 

z  —  ox  blood-cell;  a  and  b  =  partial  amboceptors  of  the  immune  sera  (a  weakly 

concentrated,  and  b  strongly  concentrated) ;   a  and  ft  =  complementophile 

groups;  ca  =  the  horse  complement  dominant  for  amboceptor  a;  c/?  =  the 

second  complement-like  constituent  of  horse  serum.     This  is  dominant 

neither  for  a  nor  for  b;   its  union,  however,  with  amboceptor  b  makes 

the  complementophile  group  a  of  amboceptor  b  capable  of  reacting. 

r  =  active  constituent  of  ox  serum  (anticomplement  amboceptor?)  which 

binds  eft. 

FIG.  3. — This  shows  the  constitution  of  the  immune  serum.     Amboceptor  a  is 

present  in  weak  concentration,  amboceptor  6  in  strong  concentration. 
FIGS.  4  and  5  illustrate  the  mechanism  of  the  haemolysis  of  strongly  prepared  ox 

blood  by  horse  serum  and  inactive  ox  serum. 

FIG.  4. — Strongly  prepared  ox  blood  is  digested  with  horse  serum.  Constituent 
eft  of  the  horse  serum  is  bound  by  amboceptor  6,  and  this  union  causes 
horse  complement  ca  to  combnie  with  amboceptor  b.  Since  ca,  however, 
is  dominant  only  for  a  and  not  for  6,  no  haemolysis  takes  place. 
FIG.  5. — Strongly  prepared  ox  blood  is  digested  with  a  mixture  of  active  horse 
serum  and  inactive  ox  serum.  Ox  serum  constituent  r  binds  component  eft 
of  the  horse  serum,  and  eft  is  thus  prevented  from  uniting  with  amboceptor  6. 
Since  the  latter,  however,  does  not  by  itself  react  with  horse  complement  ca, 
ca  is  bound  by  amboceptor  a  and  haemolysis  is  brought  about. 
FIGS.  6  and  7  illustrate  the  conditions  obtaining  when  ox  blood  is  prepared  with 
a  slight  amount  of  immune  serum,  and  when,  therefore,  only  amboceptor  b 
has  been  bound  by  the  blood-cells. 

FIG.  6. — Weakly  prepared  ox  blood  is  digested  with  horse  serum,  eft  is  bound 
by  b,  and  this  union  causes  ca  to  combine  with  b.  No  haemolysis  occurs. 
On  centrifuging,  no  horse  complement  is  found  in  the  decanted  fluids. 
FIG.  7. — Weakly  prepared  ox  blood  is  digested  with  a  mixture  of  horse  serum 
and  inactive  ox  serum.  Component  r  of  the  ox  serum  combines  with  eft. 
As  a  result  of  this  ca  is  not  bound  by  b,  and  remains  free.  On  centrifuging, 
the  decanted  fluid  contains  the  horse  complement. 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  645 


Fig.l 


Fig.2 


ox 


Fig.  6 


646  COLLECTED  STUDIES  IN  IMMUNITY. 

This,  we  believe,  disposes  of  the  objections  raised  by  Bordet 
and  Gay  against  the  view  that  in  the  haemolysis  of  guinea-pig  blood 
the  ox  serum  constituent  acts  as  an  amboceptor.  Furthermore,  a 
thorough  analysis  has  shown  that  the  interpretation  of  Bordet  and 
Gay  is  directly  opposed  to  a  number  of  experimental  observations. 
In  contrast  to  this  we  see  that  all  the  experimental  findings  fit  in 
perfectly  with  the  view  developed  on  the  basis  of  the  amboceptor 
theory.  The  peculiar  role  of  the  ox  serum  is  readily  explained  by 
the  high  affinity  of  the  complementophile  groups  which  the  serum 
contains,  or  the  high  affinity  of  the  amboceptor  to  the  complement. 
This  applies  not  only  to  the  ha3molysis  of  guinea-pig  blood,  but  also 
to  the  haemolysis  of  prepared  ox  blood.  It  is  unnecessary,  there- 
fore, to  ascribe  new  and  unique  properties  to  the  ox  serum,  as  is  done 
by  Bordet  and  Gay.  In  fact  the  apparent  exceptions  to  the  rule 
are  merely  variations  of  the  cytotoxic  action  whose  occurrence  can 
be  predicated  from  the  view  developed  on  the  basis  of  the  amboceptor 
theory. 

Resume. 

1.  Contrary  to  the  view  of  Bordet  and  Gay,  in  the  haemolysis  of 
guinea-pig  blood  by  active  horse  serum  and  inactive  ox  serum,  the 
amboceptor  is  furnished  by  the  ox  serum  and  not  by  the  horse  serum. 

2.  The  guinea-pig  blood  absorbs  the  complement  of  horse  serum 
through  the  agency  of  a  horse  amboceptor  which  is  not  dominant 
for  the  horse  complement. 

3.  Subsequent  addition  of  ox  serum  to  guinea-pig  blood  previously 
treated   with  horse   serum   does   not   produce  haemolysis,    though 
according  to  Bordet  and  Gay's  view  haemolysis  should  occur.     Neither 
is  the  haemolytic  component  of  ox  serum  thereby  bound.     This  proves 
the  incorrectness  of  Bordet  and  Gay's  theory,  according  to  which 
a  " colloid"  of  ox  serum  constitutes  a  third  element  in  the  cyto- 
toxic action,  and  is  absorbed  by  the  blood  cells  laden  with  ambo- 
ceptor and  complement,  thereby  effecting  solution  of  the  cells. 

4.  Against  this  a  direct  union  of  ox  amboceptor  and  horse  com- 
plement is  indicated  by  the  fact  that  haemolysis  is  considerably 
more  rapid  when  the  two  sera  are  digested  before  the  blood-cells 
are  added. 

5.  It  is  possible  by  treating  the  horse  serum  with  guinea-pig 
blood  at  0°  to  abstract  a  large  part  of  the  amboceptor  without 
diminishing  the  complement  content.     Despite  the  loss  of  ambo- 


JOINT  ACTION  OF  SEVERAL  AMBOCEPTORS.  647 

ceptor  the  power  of  the  horse  serum  to  produce  haemolysis  in  con- 
junction with  the  ox  serum  is  preserved.  Moreover,  when  digested 
with  blood,  such  a  serum  suffers  a  smaller  loss  of  this  power  than  does 
native  serum.  This  also  shows  that  the  amboceptor  bringing  about 
haemolysis  is  contained  in  ox  serum. 

6.  Bordet  and  Gay  found  that  ox  blood  loaded  with  amboceptor, 
(prepared),  dissolves  in  a  mixture  of  active  horse  serum  and  inactive 
ox  serum,  but  not  in  horse  serum  alone.     This  we  were  able  to  con- 
firm.    Their  interpretation,  however,   according  to   which   the   ox 
serum  acts  as  a  "  colloid  "  in  dissolving  the  ox  blood  previously  pre- 
pared with  horse  serum,  and  according  to  which  this  "colloid"  is 
bound  by  the  prepared  ox  blood,  this  interpretation  was  shown  to 
be  incorrect  for  the  following  reasons: 

7.  Prepared  ox  blood  absorbs  the  horse  complement  without 
thereby   being   dissolved.     Blood   so   treated,   however,    does   not 
dissolve  011  the  addition  of  inactive  ox  serum,  nor  has  it  the  power 
to  deprive  the  latter  of  its  ability  to  bring  about  haemolysis. 

8.  In  fact,  it  has  been  found  that  even  in  the  haemolysis  of  pre- 
pared ox  blood  inactive  ox  serum  and  horse  serum  stand  in  direct 
relations  with  each  other.     If  both  sera  are  digested  prior  to  the 
addition  of  the  prepared  ox  blood,  haemolysis  will   be   markedly 
hastened. 

9.  Ox  blood  will  also  bind  the  horse  complement  if  the  blood  is 
first  treated  with  a  small  quantity  of  amboceptor,  although  haemoly- 
sis by  horse  serum  and  inactive  ox  serum  requires  a  far  greater 
quantity    of    amboceptor.     This    shows   that   the    immune    serum 
contains  two  different  amboceptors.     One  of  these,  b,  present  in 
high  concentration,  absorbs  horse  complement  when  ox  serum  is 
absent,  the  other,  a,  present  in  weak  concentration,  binds  horse 
complement  when  ox  serum  is  present.     Only  in  the  latter  case 
does  haemolysis  occur. 

10.  Ox   serum    prevents   the  binding  of  horse  complement  by 
weakly  prepared  (amboceptor  b)  ox  blood,  and  yet  does  not  give 
rise  to  haemolysis  in  this  case. 

11.  Since,  however,  the  ox  serum  acts  on  the  horse  serum  and  not 
on  the  prepared  blood,  it  follows  that  the  ox  serum  binds  a  constituent 
of  the  horse  serum,  which  constituent  has  the  power  to  make  possible 
and  bring  about  the  union  of  the  horse  complement  and  amboceptor  b. 

12.  The  combined  action  of  the  horse  serum  and  inactive  ox  serum 
in  the  haemolysis   of  prepared  ox  blood  is  thus  explained  by  the 


648  COLLECTED   STUDIES  IN  IMMUNITY. 

anticomplementary  effect  of  the  ox  serum.  The  anticomplementary 
action,  however,  applies  in  the  main  only  to  a  complement-like 
constituent  of  horse  serum,  a  constituent  which  causes  the  effective 
horse  complement  to  unite  with  an  amboceptor  6,  although  the 
complement  is  effective  only  for  amboceptor  a.  The  phenomenon 
described  by  Bordet  and  Gay,  therefore,  cannot  be  explained  by 
their  interpretation,  whereas  all  the  experimental  data  are  easily 
understood  on  the  basis  of  the  amboceptor  theory. 


XLVII.    STUDIES    ON   ANTIAMBOCEPTORS.1 

By  C.  H.  BROWNING,  M.B.,  Ch.B.,  Glasgow,  Carnegie  Research  Fellow,  Assistant 

at  the  Institute, 

and 
Dr.  H.  SACHS,  Member  of  the  Institute. 

THE  study  of  the  antihsemolytic  effects  produced  by  immunization 
has  greatly  deepened  in  the  past  few  years  and  become  much  more 
difficult.  This  is  largely  due  to  the  recognition  of  the  complement- 
binding  power  possessed  by  albuminous  bodies  when  laden  with 
specific  antibodies.  Attention  was  called  to  this  phenomenon  by 
Gengou,2  who  concluded  that  it  demonstrated  the  existence  or 
production  of  amboceptors  against  dissolved  albuminous  bodies. 
Moreschi,3  however,  deserves  the  credit  for  first  directing  attention 
to  the  relation  of  this  phenomenon  to  the  well-known  anticomple- 
mentary  action  of  the  blood  serum.  A  study  of  Moreschi's  investi- 
gations, especially  in  the  light  of  our  present  knowledge,  makes  it 
appear  very  doubtful  whether  the  inhibiting  action  of  immune 
sera  formerly  ascribed  to  the  anticomplements  is  really  due  to  the 
presence  of  antibodies  directed  against  the  complements,  or  whether 
it  is  not  rather  occasioned,  at  least  in  a  measure,  by  the  anticomple- 
mentary  power  exerted  by  the  substance  formed  by  the  interaction 
of  albumin  and  antialbumin.  The  problem  of  differentiating  anti- 
complements  sensu  stnctiori  has  now  become  more  difficult  than  ever, 
because  the  mode  of  action  of  the  anticomplements  in  no  way  differs 
from  that  of  the  albumin  complex  laden  with  amboceptor. 

For  the  present,  therefore,  the  problem  of  demonstrating  true 
ant  ihaBmoly sins  appears  to  be  more  readily  studied  by  directing 
attention  first  to  the  antiamboceptors.  Our  knowledge  concerning 

1  Reprinted  from  the  Berlin,  klin.  Wochenschrift,  1906,  Nos.  20  and  21. 

2  Gengou,   Sur  les  sensibilatrices    des  scrums  actifs    contre  les  substances 
albuminoides.     Annales  Pasteur,  1902,  T.  XVI. 

3  Moreschi,  Zur  Lehre  von  den  Anticomplementen.     Berliner  klin.  Wochen- 
schr.,  1905.  No.  37,  and  1906,  No.  4. 

649 


650  COLLECTED  STUDIES  IN  IMMUNITY. 

the  antiamboceptors  produced  by  immunization  has  undergone 
profound  alterations  within  the  past  few  years,  thanks  to  the  funda- 
mental researches  made  by  Bordet.  These  investigations  were  fully 
confirmed  as  to  fact  by  Ehrlich  and  Sachs/  and  by  Muir  and  Brown- 
ing.2 We  must,  therefore,  assume  that  the  antiamboceptors  are 
usually  antibodies  of  the  complementophile  group,  and  in  this  respect 
must  regard  Bordet's  findings  as  a  most  conclusive  argument  in 
favor  of  the  amboceptor  theory.  Bordet's  strongest  point  consists  of 
the  fact  that  it  is  possible,  by  immunizing  with  normal  serum,  to 
produce  antiamboceptors  which  act  against  all  the  amboceptors 
(both  normal  and  immune)  of  the  species  whose  serum  was  used 
for  immunization.  But  just  this  circumstance  should  arouse  skepti- 
cism and  make  us  question  whether  perhaps  the  antiamboceptor 
effect  is  not  merely  apparent,  and  counterfeited  by  the  complement- 
binding  power  of  albumin  laden  with  antibody.  The  experimental 
analysis  of  this  case  is  far  more  easy  than  the  differentiation  of  the 
anti-complements.  In  true  antiamboceptors  the  point  of  attack 
is  a  different  one,  being  confined,  as  already  said,  to  the  comple- 
mentophile group  of  the  amboceptor.  Nevertheless,  the  differen- 
tiation of  the  antiamboceptors  is  not  as  simple  as  was  originally 
stated  in  Ehrlich  and  Morgenroth's  communications.  Suppose,  for 
example,  that  we  mix  amboceptor  and  antiamboceptor,  add  blood- 
cells,  centrifuge,  wash  the  sediment  thoroughly,  and  find,  after  the 
addition  of  complement,  that  haBmolysis  does  not  take  place.  A 
little  consideration  will  show  that  such  a  result  permits  of  two 
interpretations.  It  may  be  clue  to  an  antiamboceptor;  it  may, 
however,  be  due  to  the  complement-deflecting  power  exerted  by  an 
albuminous  precipitate  possibly  carried  down  with  the  blood-cells 
laden  with  amboceptor.  It  is  important  to  bear  in  mind  that  the 
serum  containing  the  amboceptors  also  contains  albumin  antigens, 
and  that  the  antiamboceptor  serum  contains  albumin  antibodies. 
We  fully  agree,  therefore,  with  the  statement  made  by  Pfeiffer  and 

1  Ehrlich  and  Sachs,  Ueber  den  Mechanismus  der  Antiamboceptorwirkung. 
See  page  561. 

2  Muir  and  Browning,  On  the  Properties  of  Anti-immune  bodies  and  comple- 
mentoids.     Journal  of  Hygiene,  1906,  Vol.  VI,  No.  1. 

NOTE. — Those  wishing  to  follow  the  historical  development  of  the  subject 
will  find  this  discussed  in  the  paper  by  Ehrlich  and  Sachs  already  alluded  to. 
In  this,  too,  mention  will  be  found  of  the  investigations  of  Pfeiffer  and  Fried- 
berger,  which  may  be  regarded  as  precursors  of  Bordet's  observations. 


STUDIES    ON  ANTIAMBOCEPTORS.  651 

Moreschi,1  that  "the  anticomplementary  action  of  the  precipitate 
may  counterfeit  the  existence  of  antiamboceptors." 

Using  an  amboceptor  derived  from  a  human  convalescent  from 
cholera,  Pfeiffer  and  Friedberger2  found  that  bacteriolysis  could 
be  inhibited  by  a  rabbit  serum  obtained  by  immunizing  a  rabbit 
with  human  serum.  They  concluded  from  their  experiments  that 
the  possibility  of  this  being  an  antiamboceptor  action  could  be  ex- 
cluded. It  must  be  pointed  out,  however,  that  the  results  permit 
of  another  explanation.  In  the  first  place  Pfeiffer  and  Moreschi 
believe  it  highly  improbable  that  the  antiserum  obtained  by  immun- 
izing with  normal  human  serum  should  contain  cholera  antiambo- 
ceptors. This  assumption  is  wholly  unwarranted.  We  have  already 
called  attention  to  Bordet's  observation  that  by  immunizing  with 
a  normal  serum  one  obtains  antiamboceptors  against  all  the  ambo- 
ceptors  of  the  same  species.  These  antiamboceptors,  being  directed 
against  the  complementophile  group,  are  in  their  action  entirely 
independent  of  the  cytophilic  specificity.  The  fact,  therefore,  that 
the  normal  (human)  serum  used  for  immunization  contains  no 
cholera  amboceptors,  does  not  in  any  way  argue  against  the  existence 
of  cholera  antiamboceptors. 

So  also  with  the  main  experiment  cited  by  Pfeiffer  and  Moreschi. 
This  does  not  necessarily  show  the  absence  of  antiamboceptors, 
even  though  it  does  show  the  antibacteriolytic  action  produced  by 
the  union  of  complement  and  precipitate.  Pfeiffer  and  Moreschi 
employed  an  antiserum  derived  from  rabbits  by  immunization  with 
human  serum.  When  human  cholera  serum  was  used  as  amboceptor, 
in  testing  the  precipitates  and  the  supernatant  fluids,  they  found 
that  the  precipitates  exerted  an  antibacteriolytic  action,  while  the 
supernatant  fluid  had  no  such  action.  From  the  conception  of  anti- 
amboceptors furnished  by  Bordet's  experiments,  it  might  very  well 
be  that  the  antiamboceptors  contained  in  the  antiserum  had  been 
neutralized  by  the  amboceptors  present  in  the  normal  human  serum 
used  for  precipitation.  So  far  as  the  specific  cholera  amboceptors 
are  concerned,  these  amboceptors  accordingly  have  acted  as  "anti- 
antiamboceptors,"  and  being  so  combined,  their  action  as  ambo- 
ceptors is  excluded.  All  that  can  be  claimed  for  this  experiment, 
therefore,  is  that  it  demonstrates  the  antibacteriolytic  action  of  the 


1  Pfeiffer  and  Moreschi,  Berliner  klin.  Wochenschr.  No.  2,  1906. 

2  Pfeiffer  and  Moreschi  (?) — [Translator]. 


652  COLLECTED   STUDIES  IN   IMMUNITY. 

precipitate.     It  sheds  no  light  on  the  possibility  of  antiamboceptors 
being  present  in  the  antiserum  at  the  same  time. 

The  solution  of  this  problem  is  simplified  if  we  succeed  in  excluding 
the  action  of  the  precipitate  and  so  permit  the  supposed  antiambo- 
ceptor  to  act  by  itself.  This  can  be  accomplished  by  anchoring 
the  amboceptor  to  the  cell  and  removing  the  normal  serum  constit- 
uents by  centrifuging.  From  this  point  of  view,  one  may  even  con- 
sider the  problem  as  already  solved.  The  experiments  of  Bordet, 
Ehrlich  and  Sachs,  Muir  and  Browning,  with  hsemolytic  amboceptors, 
and  those  of  Shibayama  and  Toyoda1  with  bacteriolytic  amboceptors 
all  agree  in  showing  that  the  antiamboceptor  acts  even  when  the 
cell,  loaded  with  amboceptor,  has  been  separated  from  free  serum 
constituents.  Nevertheless,  in  view  of  the  small  traces  of  albuminous 
substance  which  suffice,  when  combined  with  suitable  antibody,  to 
deflect  complement,  it  might  be  objected  that  it  is  difficult  to  com- 
pletely free  the  sedimented  blood-cells  from  traces  of  adherent 
albuminous  substances.  This  difficulty  would  appear  considerable, 
especially  if  we  incline  to  believe  that  the  blood-cells  have  some 
absorbing  action  on  the  albuminous  substances.  Furthermore, 
the  antiamboceptors  sometimes  do  not  act  at  once  on  the  amboceptor 
anchored  to  the  cell.  Bordet,  for  example,  was  unable  to  produce  the 
antiamboceptor  action  until  he  suspended  the  blood-cells  in  inactive 
serum.  This,  of  course,  diminishes  the  value  of  the  demonstration, 
since  it  introduces  a  possible  interference  due  to  complementoids 
(Muir  and  Browning) .  Our  own  observations  lead  us  to  believe  that 
the  ability  of  antiamboceptor  to  unite  with  the  amboceptor  bound  to 
the  cell  or  with  the  free  amboceptor  is  very  variable. 

In  view  of  these  objections  we  have  therefore  attempted  to  demon- 
strate the  presence  of  antiamboceptors  indirectly,  by  excluding  the 
action  of  antiamboceptors  while  allowing  antibody  and  albuminous 
substances  to  participate  in  the  reaction.  It  would  seem  that  the 
simplest  way  to  attain  this  would  be  to  employ,  as  the  source  of 
amboceptor,  a  different  species  of  animal  than  was  used  for  producing 
the  antiamboceptor. 

The  antiserum  used  by  us  was  obtained  from  a  goat  which  had 
been  immunized  with  rabbit  serum.2  The  amboceptor,  of  course, 


1  Shibayama  and  Toyoda,  Centralbl.  f.  Bact.,  Orig.  Vol.  XL,  1906. 

2  The  serum  with  which  these  animals  were  immunized  was  derived  from 
rabbits  which  had  been  treated  with  ox  blood.     It  therefore  contained  specific 


STUDIES   ON  ANTIAMBOCEPTORS. 


653 


had  to  be  derived  from  a  rabbit.  In  the  present  instance  it  was  an 
inactivated  serum  obtained  from  a  rabbit  treated  with  ox  blood. 
Guinea-pig  serum  was  used  as  complement.  In  order  to  exclude 
the  action  of  the  antiamboceptor,  a  parallel  experiment  was  made 
in  which  the  amboceptor  consisted  of  the  inactivated  serum  of  a 
goat  immunized  with  ox  blood,  guinea-pig  serum  being  used  as 
complement.  For  the  sake  of  simplicity  we  shall  term  the  ambo- 
ceptors  respectively  " rabbit  amboceptor "  and  "goat  amboceptor." 
The  experiment  is  as  follows: 

Two  series  of  test-tubes  were  prepared,  decreasing  amounts  of  the  anti- 
serum  being  placed  in  each  tube.  The  volume  in  each  tube  was  always  made 
up  to  1.0  cc.  with  physiological  salt  solution.  To  the  tubes  in  series  A  was 
then  added  0.0015  cc.  (H  solvent  doses)  of  the  rabbit  amboceptor;  while  the 
tubes  of  series  B  received  0.015  cc.  goat  amboceptor  (1£  solvent  doses)  plus 
0.0015  cc.  normal  inactive  rabbit  serum.  Both  series  of  tubes  were  kept 
at  room  temperature  for  three-quarters  of  an  hour,  after  which  1  cc.  of  a  5% 
suspension  of  ox  blood-cells  was  added  to  each  tube.  After  incubation  at  37° 
for  one  hour,  the  tubes  were  centrifuged,  the  sediments  resuspended  in  physiog- 
ical  salt  solution,  and  mixed  with  guinea-pig  serum  as  complement.  The 
amount  of  complement  also  equaled  H  solvent  doses,  being  0.075  cc.  in  seres  A, 
and  0.05  cc.  in  series  B.  After  this  the  tubes  were  kept  at  37°  for  two  hours, 
and  then  placed  in  the  refrigerator  over  night.  The  result  noted  the  next 
morning  is  shown  in  tho  following  table: 


TABLE   I. 


Amount  of  • 

Degree  of  1 

Isemolysis. 

cc. 

Series  A. 

Series  B. 

0.5 

0 

strong 

0.25 
0.15 

0 
0 

complete 

0.1 

0 

0.05 

0 

0.025 

strong 

0.015 

'  ' 

0.01 

complete 

0.005 

" 

0 

i  { 

amboceptors  for  ox  blood.  So  far  as  the  production  of  antiamboceptors  or  of 
antibodies  against  the  albuminous  substances  is  concerned,  this  is  immaterial. 
Controls  made  with  the  serum  of  a  goat  immunized  with  normal  rabbit  serum, 
yielded  the  same  results.  The  quantity  of  the  latter  available  was  too  small 
to  suffice  for  all  of  the  experiments  here  reported. 


654  COLLECTED  STUDIES  IN  IMMUNITY. 

In  order  to  analyze  the  result  of  this  experiment,  it  will  be 
advisable  to  first  have  a  clear  idea  as  to  the  .constitution  of  the 
sediments  in  the  two  series  previous  to  the  addition  of  the  comple- 
ment. In  series  A  the  sediment  consists  of: 

1.  The  blood-cells  laden  with  amboceptor. 

2.  The   antiamboceptor    (if   such   is   present   in  the  antiserum) 
bound  to  the  complementophile  group  of  the  amboceptor. 

3.  It  may  contain  the  precipitate  formed  by  the  combination  of 
albuminous  constituents  of  the  rabbit  serum  with  the  antiserum. 

In  series  B  the  sediment  also  contains  blood-cells  laden  with 
amboceptor,  but  there  is,  of  course,  no  antiamboceptor.  The  con- 
ditions for  the  formation  of  the  precipitate,  however,  are  exactly 
the  same  as  in  series  A,  for  in  both  series  the  same  quantity  of  normal 
albuminous  constitutents  of  rabbit  serum  are  present. 

In  series  B,  if  we  disregard  the  slight  inhibition  with  large  doses 
of  antiserum,  we  find  that  the  blood  cells  in  all  the  tubes  have  been 
completely  dissolved.  This  can  only  mean  that  either  the  sediments 
contained  no  precipitate,  or  that  the  precipitate  present  was  unable 
to  exert  its  deflecting  power  on  complement.  It  follows  that  the 
marked  inhibition  of  haemolysis  observed  in  series  A  must  be  ascribed 
to  the  action  of  antiamboceptor 's. 

Against  this  interpretation  it  might  be  objected  that  perhaps 
the  sediments  of  series  A  also  lack  an  antiamboceptor,  and  that  the 
inhibition  of  haemolysis  is  due  to  the  deflection  of  complement  by 
the  precipitate.  It  would  then  be  necessary  to  assume  that  the 
goat  amboceptor  possessed  a  stronger  affinity  for  the  complement 
than  did  the  rabbit  amboceptor,  in  consequence  of  which  no  deflection 
of  complement  was  produced  by  the-  precipitates  in  series  B.  In 
order  to  meet  this  objection  we  have  devised  another  experiment, 
making  use  of  the  rabbit  amboceptor  as  before,  and  excluding  the 
antiamboceptor  while  still  maintaining  the  same  favorable  con- 
ditions for  the  formation  of  a  precipitate.  The  experiment  is 
made  as  follows: 


Decreasing  amounts  of  antiserum  are  mixed  with  0.0015  cc.  inactivated 
normal  rabbit  serum,  and  the  mixtures  kept  at  room  temperature  for  forty-five 
minutes.  To  each  tube  is  then  added  1  cc.  5%  ox  blood,  the  mixtures  kept 
at  37°  for  one  hour,  and  then  centrifuged.  The  sediments  are  mixed  with 
0.0015  cc.  rabbit  amboceptor  plus  0.075  cc.  guinea-pig  serum.  It  will  be  seen 
that  the  experiment  corresponds  to  that  described  in  Table  I,  A,  except  that 
in  place  of  the  specific  amboceptor,  an  equal  volume  of  normal  serum  is  mixed 


STUDIES  ON  AXTIAMBOCEPTORS. 


655 


with  the  antiserum,  the  rabbit  amboceptor  being  added  to  the  mixture  only 
after  the  antiamboceptor  has  been  removed.  The  result  of  this  experiment 
is  shown  in  the  following  table  (Column  A  of  Table  I  may  be  regarded  as  the 

control) : 

TABLE   II. 


Amount  of  Antiserium. 
cc. 

Degree  of  Haemolysis. 

0.5 

slight 

0.25 

almost  complete 

0.15 

complete 

0.1 

0.05 

0.025 

0.015 

0 

It  will  be  seen  that  with  this  modification,  too,  the  antiserum,. 
except  in  very  large  amounts,  does  not  influence  haemolysis.  There 
can  be  be  no  doubt,  therefore,  that  the  inhibiting  factor  of  the  anti- 
serum  under  these  conditions  is  practically  only  the  antiamboceptor. 
While  these  experiments  positively  demonstrate  the  existence  of 
antiamboceptors  in  the  antiserum,  they  leave  untouched  the  question 
as  to  whether  the  antiserum  may  not  at  the  same  time  contain 
antibodies  for  albuminous  substances.  Considering  the  manner  in 
which  the  antiserum  is  produced,  it  is  natural  to  assume  that  such 
antibodies  are  formed  along  with  the  antiamboceptors.  All  that  we 
are  interested  in  at  the  present  time,  however,  is  the  possibility  of 
these  antibodies  counterfeiting  the  existence  of  antiamboceptors. 
After  the  experiments  just  described,  this  seems  out  of  the  question. 

It  might  be  doubted  whether  the  albumin  content  of  the  normal 
rabbit  serum  corresponds  to  that  of  the  rabbit  serum  specific  for 
ox  blood.  The  immune  serum  might  be  much  richer  in  albuminous 
substances.  Although  there  seems  little  basis  for  such  an  assumption, 
we  have  thought  it  advisable  to  investigate  the  matter.  In  a  further 
experiment,  therefore,  we  used  varying  quantities  of  the  normal 
rabbit  serum  with  constant  amounts  of  the  antiserum.  The  ex- 
periments were  carried  out  as  follows: 

Two  series  of  tests  are  made: 

(A)  Each  tube  contains    0.15  cc.    antiserum,  plus    0.0015  cc.   rabbit  ambo- 
ceptor, plus  decreasing  amounts  inactive  normal  rabbit  serum.     After  standing 


656 


COLLECTED  STUDIES   IN   IMMUNITY. 


forty-five  minutes  at  room  temperature,  the  ox -blood  suspension  is  added 
and  the  mixtures  kept  for  one  hour  at  37°.  After  centrifuging,  the  sediments 
are  resuspended  in  physiological  salt  solution,  and  mixed  with .0.075  cc.  guinea- 
pig  serum. 

(B)  Each  tube  contains  0.15  cc.  antiserum,  plus  decreasing  amounts  of 
inactive  normal  rabbit  serum.  After  standing  for  forty-five  minutes  at  room 
temperature,  the  ox-blood  suspension  is  added  and  the  mixtures  kept  for  one 
hour  at  37°.  After  centrifuging,  the  sediments  are  mixed  with  0.0015  cc. 
rabbit  amboceptor,  kept  at  37°  for  one  hour,  and  again  centrifuged.  To  these 
sediments  are  then  added  0.075  cc.  guinea-pig  serum. 

In  this  experiment  each  series  again  contains  the  some  con- 
stituents in  like  amounts,  the  main  difference  between  them  con- 
sisting in  the  sequence  in  which  the  constituents  are  added.  By 
having  varied  this,  we  are  enabled  to  exclude,  in  series  B,  the  action 
of  the  antiamboceptor.  The  result  of  the  experiment  is  shown  in 
Table  III. 

TABLE   III. 


Amount  of  Normal 

Degree  of  Haemolysis. 

•inactive  JXciooiL 
Serum. 

cc. 

Series  A. 

Series  B. 

0.1 

complete 

0.05 

(I 

0.025 

(  (  • 

0.015 

« 

0.01 
0.005 

strong 
moderate 

complete 

0.0025 

0 

0.0015 

0 

0.001 

0 

0 

0 

It  will  be  seen  that  despite  an  increased  amount  of  precipitable 
substance,  the  precipitate  exerts  no  binding  action  on  complement. 
In  series  A,  on  the  other  hand,  the  inhibiting  action  of  the  antiamboceptor 
is  again  very  marked.  The  experiment  also  shows  that  a  relatively 
slight  excess  of  the  normal  rabbit  serum  paralyzes  the  antiamboceptor 
action,  a  fact  which  finds  a  natural  explanation  in  the  interference 
of  the  normal  amboceptors. 

At  first  sight  the  results  shown  in  series  A  seem  somewhat  similar 
to  those  obtained  in  experiments  made  to  determine  the  amount 
of  albuminous  substance  necessary  to  produce  deflection  of  comple- 


STUDIES   ON  ANTIAMBOCEPTORS.  657 

ment  when  combined  with  the  corresponding  antiserum.  We  know 
from  the  researches  of  Fleischmann  and  Michaelis,1  as  well  as  from 
those  of  Moreschi,2  that  an  excess  of  the  albuminous  antigen  inhibits 
the  deflection  of  complement.  The  same  phenomenon  is  observed 
in  the  precipitin  reaction.  From  the  control  furnished  by  series 
B,  it  is  apparent  that  deflection  of  complement  plays  no  part  in  the 
antihffimolytic  action  noted  in  series  A.  It  follows,  therefore,  that 
the  inhibition  of  haemolysis  observed  when  large  amounts  of  serum 
are  employed,  is  to  be  regarded  as  an  antagonistic  action  exerted 
by  the  normal  serum  on  the  antiamboceptor,  and  must  be  ascribed 
to  the  normal  amboceptors  present. 

In  spite  of  this  we  may  assume  that  in  both  series  the  blood-cell 
sediments  contain  an  admixture  of  albuminous  precipitate,  for  it 
could  easily  be  shown  that  the  antiserum  possessed  precipitating 
properties.  The  serum,  to  be  sure,  was  rather  weak,  especially  so 
far  as  the  intensity  of  precipitation  was  concerned.  It  is  to  be  noted, 
however,  that  even  with  the  greatest  excess  of  rabbit  serum  occurring 
in  our  experiments,  there  was  no  failure  of  precipitate  formation; 
in  fact,  this  increased  in  proportion  to  the  amount  of  precipitable 
substance  employed.  Granted  then,  that  the  blood-cell  sediments 
contained  albumin  precipitates,  two  alternatives  may  be  offered 
to  explain  the  lack  of  deflecting  power  on  complements.  Thus,  it 
is  possible  that,  despite  the  formation  of  a  precipitate,  there  are  no 
antibodies  which  are  able  to  bind  complement,  or,  if  present,  none  that 
enter  into  the  reaction.  On  the  other  hand,  and  this  is  important 
so  far  as  the  amboceptor  problem  is  concerned,  it  is  to  be  noted  that 
with  the  technique  employed  by  us,  conditions  have  been  introduced 
which  render  occurrence  of  deflection  difficult  or  impossible.  In  order 
to  produce  deflection  of  complement,  one  proceeds  by  first  mixing 
the  albuminous  antigen,  antiserum,  and  complement,  and  subse- 
quently adding  blood  cells  and  amboceptor.  In  our  experiments, 
on  the  contrary,  the  resulting  sediment  already  contains:  1,  blood- 
cells  laden  with  amboceptor,  and  2,  the  precipitate.  The  complement 
which  is  now  added  finds  two  alternative  points  of  attachment,  and 
it  depends  entirely  on  the  relative  affinity  possessed  by  these  as  to 
where  the  complement  will  be  bound.  Had  the  complement  been 
allowed  to  react  with  the  precipitate  alone,  it  would  undoubtedly 


1  Fleischmann  and  Michaelis,  Mediz.  Klin.,  No.  1,  1906. 

2  Moreschi,  1.  c. 


658 


COLLECTED    STUDIES   IN   IMMUNITY. 


have  been  anchored,  and  then,  owing  to  the  secondary  tightening 
of  the  bonds,  would  no  longer  have  been  available  even  for  a  group 
possessing  somewhat  higher  afnnit}^  One  can  easily  convince  one's 
self  of  the  part  played  in  deflection  by  the  sequence  in  which  the 
various  reagents  are  added.  In  a  recent  study,  Michaelis  and 
Fleischmann  *  have  called  attention  to  the  sources  of  error  to  which 
disregard  of  this  circumstance  may  give  rise. 

We  made  the  following  experiment  with  our  antiserum : 

Two  series  of  tubes  were  prepared,  each  containing  0.1  cc.  antiserum  and 
decreasing  amounts  of  normal  rabbit  serum.  The  volume  was  made  up  to 
1.5  cc.  and  the  mixtures  allowed  to  stand  for  twenty-four  hours  in  order  to 
secure  the  maximum  amount  of  precipitation.  The  tubes  were  sharply  cen- 
trifuged,  and  the  supernatant  fluid  removed.  To  the  sediments  in  series  A 
were  then  added  0.075  cc.  guinea-pig  serum,  and  the  mixtures  kept  at  37°  for 
one  hour.  Then  the  ox  blood,  plus  0.0015  cc.  rabbit  amboceptor,  was  added. 
In  series  B,  the  sequence  was  altered  to:  ox  blood,  plus  0.0015  cc.  amboceptor — 
one  hour  at  37° — then  0.75  cc.  guinea-pig  serum. 

The  degree  of  haemolysis  at  the  end  of  1^  hours  is  shown  in  Table  IV. 

TABLE   IV. 


Amount  of  Normal 

Degree  of  ] 

Isemolysis. 

cc. 

Series  A. 

Series  B. 

0.25 

0 

complete 

0.15 

faint  trace 

0.1 

11 

0.05 

strong 

0.025 

0.015 

<  e 

0.01 

almost  complete 

0 

complete 

It  will  be  seen  that  the  precipitate  has  exerted  a  deflection  of 
complement,  though  not  to  a  very  high  degree;  there  is  no  deflection, 
however,  when  the  sequence  in  which  the  various  reagents  are  added 
is  the  same  as  that  employed  in  our  antiamboceptor  experiments.2 

The   essential   importance   of   the   technique    employed,    when 


1  Michaelis  and  Fleischmann,  Zeitsch.  f.  klin.  Medizin.  Vol.  58,  1906. 

2  It  is  impossible  for  us  to  say  whether  the  sequences  in  which  the  reagents 
are  added   would   have   the   same   determining   influence   when   other   ambo- 
ceptors,  especially  bactericidal  amboceptors,  are  employed. 


STUDIES  ON  ANTIAMBOCEPTORS. 


659 


making  experiments  on  the  deflection  of  complement,  was  also  well 
demonstrated  by  using  a  strong  precipitating  serum  which  had 
previously  been  employed  for  identifying  human  albumin.  This 
serum  was  obtained  from  rabbits  by  immunization  with  human 
serum,  and  is  therefore  termed  "H-R-serum."  Since  the  only 
antiamboceptors  which  this  serum  can  contain  are  those  directed 
against  human  amboceptors,  it  is  obvious  that  an  antiamboceptor 
action  is  at  once  excluded  if  we  employ  rabbit  amboceptors  specific 
for  ox  blood.  We  began  by  following  the  regular  technique  employed 
by  M.  Xeisser  and  Sachs  *  in  their  studies  on  the  forensic  blood  test 
by  means  of  antihsemolytic  action,  and  followed  this  by  two  parallel 
experiments  in  which  we  varied  the  sequence  of  the  reagents  em- 
ployed. The  details  of  the  three  tests  are  as  follows: 

Series  A.  Each  tube  contains  0.02  cc.  H-R-serum,  plus  0.05  cc.  guinea-pig 
serum,  plus  decreasing  amounts  human  serum.  Mixtures  kept  one  hour  at 
37°.  Then  1  cc.  5%  ox  blood,  plus  0.0015  rabbit  amboceptor. 

Series  B.  0.02  cc.  H-R-serum,  plus  human  serum,  plus  1  cc.  5%  ox  blood, 
plus  0.0015  rabbit  amboceptor.  After  standing  one  hour  at  37°,  0.05  cc. 
guinea-pig  serum. 

Series  C.  0.02  cc.  H-R-serum,  plus  human  serum,  plus  1  cc.  5%  ox  blood, 
plus  0.0015  rabbit  amboceptor.  After  standing  for  If  hours  at  37°,  the 
mixtures  are  centrifuged.  Then  0.05  guinea-pig  serum  is  added  to  the  sedi- 
ments. 

In  series  C,  the  mixtures  were  kept  at  37°  for  If  hours  in  order  to  furnish 
more  favorable  conditions  for  the  formation  of  a  precipitate,  and  also  so  that 
the  conditions  as  to  time  would  be  the  same  as  those  in  the  antiamboceptor 
experiment.  In  series  B  and  C,  the  guinea-pig  serum  was  kept  at  37°  for 
one  hour  previous  to  mixing. 

The  result  of  this  experiment  is  shown  in  Table  V. 


TABLE  V. 


Amount  of  Human 

Degree  of  Haemolysis. 

cc. 

Series  A. 

Series  B. 

Series  C. 

0.001 

0 

trace 

0.0001 

0 

<  < 

0.00001 
0.000001 
0 

0 
0 

complete 

moderate 
complete 

complete 

1  Xeisser  and  Sachs,  Berliner  klin.  Wochenschrift,  No.  3,  1906. 


660  COLLECTED  STUDIES  IN  IMMUNITY. 

In  series  A  we  see  deflection  of  complement  very  well  marked; 
in  series  B,  in  which  complement  was  added  last,  the  deflection  is 
considerably  lessened,  and  when  the  additions  are  made  in  accord- 
ance with  the  technique  of  the  antiamboceptor  experiment,  we  find 
that  there  is  no  deflection  whatever.  In  this  experiment,  as  already 
explained,  we  made  use  of  an  antiserum  having  strong  precipitating 
and  deflecting  power.  The  result  confirms  our  contention  that 
the  inhibiting  action  observed  in  our  previous  experiments  is  not 
due  to  the  formation  of  a  precipitate,  but  is  caused  solely  by  anti- 
amboceptors. 

Even  when  present,  the  precipitates  are  unable  to  exert  a  deflec- 
tion on  the  complement  provided  blood-cells  laden  with  amboceptor 
.are  present  at  the  same  time,  so  that  the  complement  subsequently 
introduced  has  the  alternative  of  combining  with  precipitate  or  with 
the  prepared  blood-cells.  At  the  same  time  we  must  call  attention 
to  a  possibility  which  makes  it  likely  that  an  intensification  of  the 
power  of  the  precipitate  occurs  in  connection  with  the  antiamboceptor 
action.  Conditions  might  exist  under  which  the  complement  would 
replace  the  antiamboceptor  already  bound  to  the  amboceptor,  were 
not  the  precipitate  present  at  the  same  time.  It  is  possible  that 
this  explains  the  varying  results  obtained  in  attempts  to  definitely 
replace  with  antiamboceptor  the  normal  amboceptor  already  anchored 
to  the  cell,  and  freed  from  normal  serum  constituents.1  It  is  con- 


1  Ehrlich  and  Sachs  (I.e.)  called  attention  to  a  paradoxical  phenomenon, 
which  consisted  in  the  fact  that  the  sensitized  blood-cells  were  protected  only 
by  small  doses  of  the  antiserum,  while  an  excess  of  antiserum  did  not  inhibit 
haemolysis.  They  found,  however,  that  the  antiha3molytic  effect  was  produced 
even  with  an  excess  of  antiserum,  provided  a  small  quantity  of  normal  serum 
homologous  to  the  amboceptor  was  added.  Moreschi  (I.e.)  interprets  this 
as  indicating  an  anticomplementary  action  due  to  the  formation  of  a  pre- 
cipitate. In  opposition  to  this,  it  may  be  remarked  that  under  analogous 
conditions  the  formation  of  a  precipitate  does  not  lead  to  a  deflection  of  com- 
plement. The  peculiarity  of  the  phenomenon  described  by  Ehrlich  and  Sachs 
consists  not  alone  in  the  fact  that  the  antiserum  acts  only  after  the  addition 
of  normal  serum.  The  striking  thing  is  that  an  excess  should  cause  the  anti- 
serum  to  lose  its  inhibiting  property.  In  this  the  presence  and  coaction  of 
normal  serum  constituents  (precipitable  substances)  are  entirely  out  of  the 
question.  Hence,  while  at  first  sight  Moreschi's  explanation  appears  very 
apt,  we  see  that  it  is  insufficient  to  throw  light  on  the  entire  group  of  facts 
presented  by  Ehrlich  and  Sachs.  For  the  present  it  will  be  difficult  to  get 
along  without  accepting  the  possibility  suggested  by  those  authors,  namely, 


STUDIES  ON^  ANTIAMBOCEPTORS.  661 

ceivable  that  even  under  these  circumstances,  the  antiamboceptor 
is  always  bound,  but  that  in  many  cases  this  union  can  still  be 
dissolved  by  the  complement  owing  to  the  absence  of  the  deflecting 
precipitate.  If  we  accept  this  secondary  participation  of  the  pre- 
cipitate in  the  antiamboceptor  action,  it  is  easy  to  understand  the 
apparent  failure  of  the  antiamboceptor  to  be  bound  to  the  sensitized 
blood-cells.  Some  explanation  for  this  lack  of  combination  is 
certainly  desirable.  One  would  naturally  expect  the  antiambo- 
ceptor to  act  more  powerfully  on  the  sensitized  blood-cells,  for  in 
blood-cells  laden  with  amboceptor  the  free,  normal  amboceptors  of 
the  immune  serum  are  absent.  These  free  amboceptors  come  into 
action  when  the  antiamboceptor  acts  directly  on  the  entire  immune 
serum,  and  they  can  thus  lower  the  action  of  the  antiamboceptor  on 
the  specific  amboceptor.  As  a  matter  of  fact,  we  have  encountered 
instances  in  which  the  antiserum  acted  just  as  strongly  on  the 
sensitized  cell  as  on  the  native  immune  serum.  In  other  cases, 
however,  the  antiserum,  when  employed  in  accordance  with  the 
usual  technique  (sensitized  blood  +  antiserum — one  hour  at  37°— 
centrifuging — addition  of  complement — two  hours  at  37°),  exerted 
no  action  whatever.  This  was  the  case  with  the  antiserum  whose 
properties  we  have  discussed  in  this  paper. 

These  considerations  led  us  to  see  if  we  could  make  the  action 
of  the  antiserum  on  the  sensitized  cell  visible.  To  do  this  we  felt 
that  two  things  in  particular  had  to  be  regarded.  In  the  first  place, 
it  seemed  advisible  to  leave  the  antiserum  in  contact  with  the  blood- 
cells  laden  with  amboceptor  as  long  as  possible,  in  order  to  effect 
the  maximum  amount  of  binding  with  the  antiamboceptor.  This 
would  make  it  more  difficult  for  the  complement  subsequently  added 
to  dislodge  the  antiamboceptor.  In  the  second  place,  it  seemed 
probable  that  the  complement  only  gradually  displaced  the  anti- 
amboceptor, and  that  examinations  made  at  intervals  would  reveal 
a  phase  in  which  an  antiamboceptor  action  can  be  observed. 

We  arranged  our  experiment  as  follows: 


an  interfering  action  produced  by  two  antibodies  in  the  antiserum,  bodies 
having  the  type  of  antiamboceptors.  So  far  as  the  details  are  concerned  we 
must  refer  to  the  original  paper  of  Ehrlich  and  Sachs.  Here  we  would  only 
remark  that  the  interpretation  given  at  that  time  is  applicable  also  to  those 
cases  in  which  the  antiamboceptor  is  without  effect  when  sensitized  blood- 
cells  freed  from  normal  serum  constituents  are  employed. 


662 


COLLECTED  STUDIES  IN  IMMUNITY. 


To  1  cc.  5%  ox  blood  are  added  0.0015  cc.  rabbit  amboceptor,  and  the 
mixtures  kept  at  37°  for  one  hour.  Each  tube  receives  decreasing  amounts 
of  antiserum,  those  in  series  A  directly,  and  those  in  series  B,  to  the  blood- 
cells  separated  by  centrifuge  and  freed  from  the  fluid  medium  in  which  they 
had  been  suspended.  The  two  series  therefore  contained,  in  addition  to  the 
antiserum: 

Series  A.  Blood-cells  laden  with  amboceptor,  plus  free  normal  ambocep- 
tors,  plus  precipitable  substance. 

Series  B.  Only  blood-cells  laden  with  amboceptor. 

Both  sets  of  tubes  are  kept  at  37°  for  two  hours,  then  in  the  refrigerator 
over  night,  and  centrifuged  the  next  morning.  The  sediments  are  suspended 
in  physiological  salt  solution  to  which,  for  each  tube,  1£  solvent  doses  of 
guinea-pig  serum  have  been  added  (0.03  cc.).  The  degree  of  haemolysis  is 
noted  at  the  end  of  J  and  2  hours.  See  Table  VI. 


TABLE   VI. 


Degree  of  Haemolysis. 

Amount  of 

Antiserum. 

Series  A. 

Series  B. 

cc. 

After  $  Hour. 

After  2  Hours. 

After  £  Hour. 

After  2  Hours. 

0.25 

0 

0 

0 

moderate 

0.15 

0 

0 

0 

<  < 

0.1 

0 

faint  trace 

0 

(  ( 

0.05 

faint  trace 

strong 

0 

strong 

0.025 
0.015 

trace 
moderate 

complete 

0 
0 

1  1 
almost  complete 

0.01 

almost  complete 

moderate 

complete 

0.005 

1  1 

(  i 

« 

0.0025 

complete 

t( 

i  ( 

0.0015 

i  ( 

strong 

1  1 

0.001 

1  1 

(i 

(  t 

0 

(i 

complete 

1  1 

A  number  of  points  are  brought  out  by  this  table.  In  series  B  we  observe 
that  the  antiamboceptor  has  exerted  a  distinct  influence  on  the  antiambo- 
ceptor l  anchored  by  the  cells  and  freed  from  other  serum  constituents. 
Examining  the  tubes  at  the  end  of  half  an  hour  we  see  that  haemolysis  has 
been  markedly  inhibited.  Subsequently,  however,  this  inhibition  gradually  dis- 
appears, so  that  at  the  end  of  two  hours  what  little  antihsemolytic  action  is 
still  present  is  insignificant  when  compared  to  the  antiamboceptor  action  at 
the  end  of  half  an  hour.  This  result  agrees  very  well  with  the  assumption  that 
the  complement  is  able,  after  a  time,  to  dislodge  the  antiamboceptor.  On 
comparing  the  results  in  series  B  with  those  in  series  A,  we  note  that  the 


Misprint  for  amboceptor  (?)— [Editor.] 


STUDIES  ON  AXTIAMBOCEPTORS.  663 

inhibition  of  haemolysis  at  the  end  of  half  an  hour  is  less  marked  in  the  latter. 
One  would  have  expected  the  contrary  to  be  the  case,  or  the  presence  of  pre- 
cipitable  substance  in  series  A  furnishes  conditions  favorable  to  the  formation 
of  a  precipitate.  It  must  not  be  forgotten,  however,  that  the  mixtures  in 
series  A  also  contain  free  normal  amboceptors  (eliminated  in  series  B)  and 
these  may  be  able  to  diminish  the  antiamboceptor  action.  This  is  all  the 
more  likely  since  these  amboceptors  are  free  in  solution  and  therefore  more 
readily  able  to  react  with  the  antlamboceptors  than  are  the  specific  am- 
boceptors already  bound  to  the  cell. 

At  the  end  of  two  hours,  on  the  other  hand,  we  find  that  the  antiambo- 
ceptor action  is  more  marked  in  series  A  than  in  series  B.  On  the  basis  of 
the  above  assumption,  this  might  be  due  to  the  fact  that  the  precipitate 
produced  by  the  large  quantities  of  antiserum  is,  in  a  way,  a  deflector  of 
complement,  since  it  robs  the  complement  of  its  tendency  to  break  up  the 
amboceptor-antiamboceptor  combination.  Under  the  conditions  obtaining, 
the  complement-binding  power  of  the  precipitate  is  too  small  to  prevent  the 
complement  uniting  with  the  free  complementophile  group  of  the  amboceptor, 
but  is  large  enough  to  restrain  it  when  the  complementophile  group  is  already 
occupied  by  the  antiamboceptor.  Precipitate  and  antiamboceptor  would  thus 
at  times  mutually  support  each  other  in  their  action. 


To  what  extent  such  a  combined  action  really  occurs  must  be 
left  to  future  investigations.  In  any  case,  we  believe  it  important 
to  bear  this  possibility  in  mind,  in  order  to  gain  a  clear  idea  of  all 
the  conditions  which  may  play  a  part  in  the  action  of  anti- 
amboceptors. 

Each  of  the  two  factors  (precipitate  and  antiamboceptor)  will 
surely  also  be  able  to  exert  an  antihamolytic  effect  by  itself.  The 
independent  action  of  the  antiamboceptor  is  demonstrated  further 
by  the  fact  that  it  persists  even  when  the  complement  is  increased 
several  times.  If  the  inhibition  were  due  only  to  precipitates,  we 
should  expect  that  it  would  be  overcome  by  an  excess  of  complement, 
since  the  precipitate  acts  only  as  an  anticomplement.  On  the  con- 
trary, it  can  be  shown  that  the  inhibition  produced  by  the  anti- 
amboceptor persists  even  when  the  dose  of  complement  is  consider- 
ably increased.  It  might  be  thought  that  a  precipitate  present  at  the 
same  time  binds  all  the  complement  added,  but  this  is  not  the  case. 
It  is  possible  to  demonstrate  the  presence  of  sufficient  free  complement 
by  separating  the  fluid  from  the  undissolved  blood-cells,  and  allowing 
it  to  act  on  native,  sensitized  blood-cells.  This  fact  agrees  with 
Bordet's  observation,  that  the  antiamboceptor  robs  sensitized  blood- 
cells  of  the  power  to  bind  complement.  When  we  employed  a 
very  small  quantity  of  complement,  just  sufficient  to  produce  com- 


664 


COLLECTED  STUDIES  IN  IMMUNITY. 


plete  haemolysis,  we  did,  to  be  sure,  observe  a  slight  loss  of  comple- 
ment, despite  the  presence  of  antiamboceptor.  We  believe  that  this 
is  caused  by  the  presence  of  a  very  small  amount  of  precipitate.  The 
important  fact,  however,  is  that  we  could  demonstrate  plenty  of 
free  complement,  although  there  was  no  haemolysis.1  A  brief  de- 
scription of  such  an  experiment  follows: 

To  0.125  cc.  antiserum  are  added  0.0015  cc.  rabbit  amboceptor, 
and  the  mixtures  kept  at  room  temperature  for  45  minutes.  Ox 
blood  is  added,  and  the  mixtures  kept  at  37°  for  one  hour.  After 
centrifuging,  the  sediments  are  mixed  with  guinea-pig  serum,  as 
follows : 

1.  0.075  cc.  =  l^  complete  solvent  doses. 

2.  0.1      cc.  =  2    solvent  doses. 

3.  0.2      cc.  =  4    solvent  doses. 

4.  0.3      cc.  =  6  solvent  doses. 

The  tubes  are  kept  at  37°  for  two  hours,  then  over  night  in  the 
ice  chest.  The  following  day  the  supernatant  fluids  are  carefully 
poured  off  and  tested  for  complement  by  adding  the  sediments 
obtained  from  1  cc.  5%  ox  blood  plus  0.0015  cc.  rabbit  amboceptor. 
The  result  is  shown  in  Table  VII. 


TABLE  VII. 


Amount  of 
Guinea-pig  Serum, 
cc. 

Degree  of  Haemolysis. 

(a) 
Of  the  Original 
Mixtures. 

(6) 
Of  the  Decanted 
Fluids  Digested 
with  Ox  Blood  plus 
Amboceptor. 

0.075 
0.1 
0.2 
0.3 

0 
0 
0 
0 

strong 

complete 

n 

K 

Although  as  indicated  in  the  first  column  of  the  table,  there  is  a 
moderate  diminution  of  complement,  we  note  that  despite  a  plentiful 
amount  of  complement,  haemolysis  does  not  occur.  The  reason  for 


1  We  see,  therefore,  that  the  ability  of  the  complement  to  dislodge  anchored 
antiamboceptor  (if  such  a  power  is  at  all  possessed  by  the  complement)  does 
not  always  manifest  itself. 


STUDIES  ON  ANTIAMBOCEPTORS.  665- 

this  is  because  the  complementophile  group  of  the  amboceptor  i& 
occupied  by  the  antiamboceptor,  whereby  this  point  of  attachment  is 
blocked  for  the  complement  as  with  a  complementoid. 

Summing  up  the  results  of  our  experiments,  we  must  conclude 
that  it  is  impossible  longer  to  doubt  the  existence,  in  the  antiserum,. 
of  antibodies  directed  against' hsemolytic  amboceptors.  It  is  possible 
to  differentiate  them  in  their  action,  even  when  antibodies  for 
albuminous  substances  are  present  at  the  same  time.  This  estab- 
lishes the  antiamboceptors  as  inhibiting  substances  sui  generis. 
By  the  formation  of  precipitates,  the  albumin-antibodies  may,  at 
times,  more  or  less  favor  the '  action  of  the  antiamboceptors,  without,, 
however,  exhibiting  the  complement-binding  power  inherent  in  them. 


XLVIII.    DISSOCIATION  PHENOMENA  IN  THE  TOXIN- 
ANTITOXIN  COMBINATION.1 

By  Doctors  R.  OTTO  and  H.  SACHS. 

» 

IN  recent  years  a  number  of  investigators  have  called  attention 

to  a  curious  paradoxical  phenomenon,  namely,  that  with  suitable 
mixtures  of  toxin  and  antitoxin  the  toxicity  for  animals  is  the  greater 
up  to  a  certain  point,  the  smaller  the  fractional  part  injected.  It  is 
to  the  keen  observation  of  Behring2  that  we  owe  the  first  data  on 
this  subject.  Behring  found  that  the  injection  of  1-50,  or  even  1-500, 
part  of  a  mixture  of  tetanus  toxin  and  tetanus  antitoxin  was  more 
highly  toxic  for  mice  than  the  injection  of  the  entire  amount.  It 
should  at  once  be  stated  that  the  fractional  parts  were  diluted  with 
water,  so  that  the  volume  injected  was  the  same  in  all  cases.  Analo- 
gous observations  were  recently  made  by  Madsen  3  working  with 
the  toxin  of  botulism.  This  investigator  found  that  toxin-antitoxin 
mixtures  which  exerted  only  very  slight  toxic  effects  might  still  kill 
guinea-pigs,  if  but  the  fortieth  or  eightieth  part  of  the  mixture  were 
used.  Similarly,  the  slight  toxic  effects  of  the  full  amount  could 
be  entirely  avoided  if  ten  times  this  quantity  was  injected.  We,  too, 
encountered  the  phenomenon  some  years  ago  in  the  course  of  test 
tube  experiments  on  the  hsemolytic  action  of  garden-spider  toxin. 
After  the  publication  of  Madsen's  observations,  we  took  up  the 
question  anew,  and  studied  the  phenomenon  in  mixtures  of  botulism 
toxin  and  antitoxin.  In  view  of  the  interest  which  attaches  to  the 


1  Reprinted  from  Zeitschr.  f.  exp.  Pathol.  u.  Therapie,  Vol.  Ill,  1906. 

2  E.  von  Behring,  Aetiologie  and  aetiologische  Therapie  des  Tetanus.     Beh- 
ring's  Beitrage  zur  experimentellen  Therapie,   Heft  7,  1904,  p.  51;    also  ibid. 
Heft  3,  1900,  p.  1092. 

3Th.  Madsen,  Gifte  und  Gegengifte,  Centralblatt  f.  Bacteriologie,  Referate, 
Vol.  37,  1905;  also  Proceedings  of  the  Danish  Academy  of  Sciences,  Meeting, 
Dec.  16,  1904. 

666 


DISSOCIATION  IX  THE  TOXIX-AXTITOXIX  COMBIXATION.      667 

subject,  we  have  felt  it  advisable  to  publish  the  results  of  our  experi- 
ments, especially  since  they  shed  some  light  on  the  cause  of  the 
paradoxical  results. 

The  botulism  toxin  and  its  antitoxin  was  kindly  furnished  us  by 
Professor  Forssmann  of  Lund.  We  began  by  experimenting  with 
mice,  and  first  determined  the  lethal  dose  by  subcutaneous  injections. 
This  is  shown 'in  the  following  table: 


TABLE  I. 


Dose  of  Toxin. 

Effect  on  the  Animal. 

Remarks. 

0.0002 
0.0001 
0.00009 
0.00008 

f2* 

t3 

lives 

sick  8  days 
sick  3  days 

*  t2,  etc.,  denote  death  on  the  second  day,  etc. 

We  next  determined  the  L1"  quantity  of  the  antitoxin,  using 
1,000  times  the  fatal  dose  (0.1  cc.)  for  this  purpose.  The  mixtures 
of  toxin-antitoxin  were  allowed  to  stand  for  three  hours  at  room 
temperature  previous  to  injection.  The  result  of  this  test  is  shown 
in  Table  II. 

TABLE  II. 


Dose  of  Toxin. 

Dose  of 
Antitoxin. 

Effect  on  the 
Animals. 

Remarks. 

0. 

0.001 

lives 



0. 

0.0009 

<  < 

— 

0. 

0.0008 

tt 

— 

0. 

0.0007 

(  C 

sick  1  day 

0. 

0.0006 

(  ( 

sick  4  days 

0. 

0.0005 

t4 

— 

0. 
0. 

0.0004 
0.0003 

1? 

— 

The  experiments  proper  began  with  a  mixture  of  toxin,  0. 1  + 
antitoxin,  0.0006.  The  mice  were  injected  subcutaneously  with 
1/1,  1/2,  1/5,  1/10,  etc.,  of  this  mixture.  The  dilutions  were  pre- 
pared immediately  before  the  injection,  and  the  volume  of  the  fluid 
injected  was  always  1  cc.  The  result  is  shown  in  Table  III. 


668 


COLLECTED  STUDIES  IN  IMMUNITY. 
TABLE   III. 


The  Injection  was  Made 

Fractional  Part 
of  the  Mixture 

A 

g 

(0.1  Toxin  + 
0.0006  Anti- 

Directly after  Mixing. 

After  Three  Hours'  Standing. 

toxin)  Injected 

Effect. 

Remarks. 

Effect. 

Remarks. 

1/1 

1/2 

lives 

i  t 

sick  2  days 
sick  4  days 

lives 

(  t 

sick  3  days 
sick  3  days 

1/5 

•2 

— 

sick  5  days 

1/10 

1* 

— 

t3 

— 

1/20 

? 

— 

t3 

— 

1/50 

\2 

— 

t5 

— 

1/75 

•r2 

— 

t4 

— 

1/100 

t3 

•"  • 

lives 

sick  2  days 

The  table  needs  no  further  explanation.  It  completely  confirms 
the  results  obtained  by  Madsen,  and  exhibits  the  paradoxical  phe- 
nomenon in  the  clearest  manner.  It  should  be  noted  that  it  makes 
very  little  difference  whether  the  dilutions  of  the  original  mixture 
and  the  injections  are  made  immediately  after  preparing  the  mixture 
or  after  the  latter  has  stood  for  three  hours,  though  the  phenomenon 
is  perhaps  somewhat  more  striking  if  the  injections  are  made  at 
once. 

A  deeper  insight  was  afforded  when  we  used  rabbits  for  the 
inoculations,  for  then  we  were  able  to  apply  the  toxin-antitoxin 
mixtures  by  means  of  intravenous  injections.  A  comparison  of  the 
L1"  values  in  rabbits,  both  with  subcutaneous  and  intravenous 
injections,  at  once  showed  marked  differences.  Thus  when  we 
injected  toxin-antitoxin  mixtures 'which  had  stood  three  hours,  we 
found  the  intravenous  injections  to  be  considerably  more  toxic  than 
the  subcutaneous.  If,  however,  we  waited  24  hours  after  preparing 
the  mixtures,  and  then  injected,  we  found  that  this  difference 
was  practically  wiped  out.  Such  an  experiment  is  reproduced  in 
Table  IV. 

From  the  table  we  see  that  the  toxicity  of  the  mixtures  by  sub- 
cutaneous injection  has  been  but  slightly  altered  by  the  24  hours7 
standing;  there  is  perhaps  a  little  impairment,  but  it  is  inconsider- 
able. When  intravenous  injections  are  employed,  however,  a  marked 
loss  of  toxicity  is  caused  by  the  twenty-four  hours'  standing.  In  the 
case  of  this  botulism  toxin  we  are  apparently  dealing  with  the  same 
conditions  which  Morgenroth  has  described  in  the  case  of  diphtheria 


DISSOCIATION  IN  THE  TOXIN-ANTITOXIN  COMBINATION.      669 

TABLE   IV. 
(A)   DETERMINATION  OF  Lf  WHEN  MIXTURES  HAVE  STOOD  3  HOURS. 


Dose  of 
Toxin. 

Dose  of 
Antitoxin. 

Subcutaneous. 

Intravaneous. 

Effect. 

Remarks. 

Effect. 

Remarks. 

0.1 
0.1 
0.1 

0.0004 
0.0007 
0.001 

t4 
lives 

sick  2  days 

t2 

t3 

t3 

— 

(B)  DETERMINATION  OF  Lf  WHEN  MIXTURES  HAVE  STOOD  24  HOURS. 


0.1 

0.-0001 

t3 



t2 



0.1 

0.0002 

1"4 

— 

"M 

— 

0.1 

0.0004 

f!7 

— 

tie 

— 

0.1 
0.1 

0.0007 
0.001 

lives 

lively 

lives      < 
lives 

slightly  ill 
3  days 
lively 

toxin.  Morgenroth  *  found  that  the  reaction  between  diphtheria 
toxin  and  its  antitoxin  proceeded  slowly,  but  that  the  time  relations 
could  be  brought  out  only  by  maens  of  intravenous  injections.  When 
subcutaneous  injections  were  employed,  the  length  of  time  which 
the  toxin-antitoxin  mixtures  remained  in  contact  appeared  to  have 
no  influence  whatever.  Morgenroth  therefore  assumed  "  that  in  the 
subcutaneous  areolar  tissue  certain  factors  are  present  which  hasten 
the  union  of  toxin  and  antitoxin."  His  idea,  then,  is  that  the  reac- 
tion is  hastened  by  certain  positive  catalytic  influences.2  We  shall 
probably  not  err  if  we  interpret  our  own  results,  with  botulism  toxin, 
in  the  same  manner,  and  assume  that  they  are  the  result  of  a' slow 
reaction  between  toxin  and  antitoxin,  which  reaction  is  hastened  in 
the  subcutaneous  connective  tissue. 

In  view  of  these  facts  one  might  assume  that  the  increased  tox- 
icity  of  fractional  portions  of  a  relatively  neutral  toxin-antitoxin 
mixture  was  due  to  the  catalytic  action  of  the  tissues,  somewhat  in 


1  Morgenroth,  Untersuchungen  iiber.  die  Bindung  von  Diphtherietoxin  und 
Antitoxin,  zugleich  ein  Beitrag  zur  Kenntniss  der  Constitution  des  Diphtherie- 
giftes.      Zeitschrift    f.     Hygiene,    Vol.     XL VIII,    1904;     also    Berliner    klin. 
Wochenschr.,  No.  20,  1904. 

2  Attention  may  be  called  to  the  fact  that  von  Behring  assumed  the  exist- 
ence of  a  positive  katalysator   (conductor)   in  fresh  tetanus  antitoxin.     See 
Deutsche  med.  Wochenschrift,  No.  35,  1903. 


670 


COLLECTED  STUDIES  IN  IMMUNITY. 


the  following  manner:  the  original  mixture  injected  subcutaneously 
is  not  yet  completely  neutralized  and  becomes  so  only  through  the 
catalytic  action  of  the  subcutaneous  tissues.  It  is  conceivable  that 
this  catalytic  action  might  become  less  with  decreasing  concentra- 
tion of  the  toxin-antitoxin  mixture,  so  that  the  original  concentrated 
solution  proved  non-poisonous  while  a  fractional  part  of  the  same, 
through  the  absence  of  the  neutralizing  catalytic  action,  would  still 
be  toxic.  Such  an  assumption  would  at  least  explain  certain  of  the 
observed  facts.  It  seemed  advisable,  therefore,  to  repeat  the  ex- 
periments in  such  a  way  as  to  exclude  the  catalytic  action  of  the 
subcutaneous  tissue  and  this  was  easily  possible  by  injecting  rabbits 
intravenously.  The  determinations  of  the  L^  dose  for  rabbits  are 
shown  in  the  following  table: 


TABLE  V. 


Intravaneous  Injection  after 

Amount  of 
Toxin. 

Amount  of 
Antitoxin. 

Standing  3  Hours. 

Standing  24  Hours. 

Result. 

Remarks. 

Result. 

Remarks. 

0.5 

0.001 

— 

— 

t2 



0.5 

0.0015 

— 

— 

t3 

— 

0.5 

0.002 

f2 

— 

'  t6 



0.5 

0.003 

f4 

— 

lives 

ill  2  days 

0.5 

0.004 

fl5 

— 





0.5 

0.005 

lives 

lively 

— 

— 

Having  obtained  these  data,  we  injected  two  series  of  rabbits  with 
dilutions  of  the  following  mixtures: 

(a)  0.5  toxin  plus  0.004  antitoxin  standing  3  hours. 

(6)  0.5  toxin  plus  0.003  antitoxin  standing  24  hours. 

'the  result  of  the  experiment  is  shown  in  Table  VI. 

From  this  table  we  see  at  once  that  even  when  intravenous  injec- 
tions are  employed,  the  increased  toxicity  of  fractional  portions  of 
toxin-antitoxin  mixtures  is  still  strikingly  manifested.  We  shall, 
therefore,  have  to  assume  that  really  neutralized  mixtures  of  toxin 
and  antitoxin  become  more  toxic  on  dilution,  that,  in  other  words, 
there  is  a  dissociation  of  the  toxin-antitoxin  combination  when  the 
mixtures  are  diluted.  From  Table  VI6,  moreover,  we  learn  that 
this  dissociability  almost  disappears  when  the  mixtures  have  stood. 


DISSOCIATION  IN  THE  TOXIN-ANTITOXIN  COMBINATION.      671 

TABLE   VI. 


Fractional 

Mixture  a. 

Mixture  6. 

Portion  of  the 

Mixture 

Injected. 

Result. 

Remarks. 

Result. 

Remarks. 

1/1 

lives 

ill  2  days 

lives 

well(?) 

1/2 

t5 

- 

14 

1/4 

ts 

— 

11 

ill  a  long  time 

1/8 

•    lives 

ill  4  days 

tl 

well(?) 

1/16 
1/32 

lives 
lives 

well 

tt 
1  1 

ill  several  days 
well 

for  some  time.  With  mixtures  that  have  stood  24  hours  before  dilut- 
ing, there  is  practically  no  increase  in  toxicity  as  a  result  of  dilution, 
and  this  is  all  the  more  noticeable  because  the  mixture  which  stood 
24  hours  contained  only  three-quarters  of  the  quantity  of  toxin  con- 
tained in  that  which  stood  only  three  hours.  It  is  necessary,  there- 
fore, to  distinguish  two  phases  in  the  reaction  between  toxin  and 
antitoxin,  a  primary  phase  in  which  neutralization  has  taken  place, 
but  in  which  dilution  suffices  to  again  liberate  some  of  the  toxin,  and 
a  secondary  phase  in  which  this  is  no  longer  possible  or  is  possible 
only  to  a  very  slight  degree.  The  assumption  of  these  two  phases 
accords  completely  with  Ehrlich's  views  concerning  the  relations 
existing  between  toxin  and  antitoxin.  We  assume  that  in  the  toxin- 
antitoxin  reaction  there  exists  a  stage  in  which  the  reaction  is  to  a 
certain  extent  reversible,  and  that  this  is  succeeded  by  a  tightening 
of  the  bonds,  a  stage  of  firm  union,  in  which  the  reversibility  is  lost. 
The  most  striking  example  of  this  secondary  tightening  is  that  known 
as  the  Danysz-Dungern  1  phenomenon,  which  consists  in  the  demon- 
stration of  increased  toxicity  of  toxin-antitoxin  mixtures  by  the 
fractional  additional  of  the  toxin. 

In  the  phenomenon  which  we  are  studying,  the  first  stage  of  the 
reaction,  namely,  that  of  reversibility,  is  brought  out  by  diluting  the 
mixtures.  It  has,  of  course,  long  been  known  that  the  union  of 
toxin  and  antitoxin  proceeds  more  rapidly  in  concentrated  than  in 
dilute  solutions,  and  this  has  from  the  outset  been  emphasized  by 
Ehrlich.  WTiat  was  new  about  these  observations  was  the  fact  that 
neutralized,  concentrated  toxin-antitoxin  mixtures  could  be  disso- 


1  v.  Dungern,  Deutsch.  med.  Wochenschr.,  1904;   Sachs,  Berl.  klin.  Wochen- 
schr.,  1904;  and  Centralbl.  Bacteriol.  I  Abt.,  Orig.  Vol.  XXXVII,  1904. 


672  COLLECTED  STUDIES  IN  IMMUNITY. 

ciated  to  so  great  a  degree  by  diluting  the  mixtures.  The  process 
reminds  one  in  a  way  of  the  well-known  chemical  phenomenon  of 
hydrolytic  dissociation.  To  mention  but  a  single  example,  acetic 
acid  and  alcohol  unite  to  form  ethylacetate.  Conversely,  however, 
when  diluted  with  water,  ethylacetate  decomposes  into  its  two 
components,  acetic  acid  and  alcohol.  If  we  regard  the  toxin  as  the 
acetic  acid,  the  antitoxin  as  the  alcohol,  and  the  neutral  toxin-anti- 
toxin mixture  as  the  product  of  the  two,  ethylacetate,  we  get  a  good 
picture  of  what  occurs  when  we  dilute  the  toxin-antitoxin  mixture. 
Our  experiments  show,  then,  that  by  diluting  neutral  toxin-antitoxin 
mixtures  it  is  possible  to  recover  the  two  components,  toxin  and  anti- 
toxin, up  to  a  certain  point.  Furthermore,  the  possibility  of  doing 
this  by  dilution  exists  for  only  a  comparatively  short  time.  After 
this  the  secondary  tightening  of  the  bonds  effects  such  a  firm  union 
that  this  mode  of  separating  the  two  components  does  not  avail. 
By  making  use  of  special  methods,  however,  it  is  possible,  even  after 
a  considerable  time,  to  liberate  the  toxin  from  a  neutral  toxin-anti- 
toxin mixture.  This  is  well  shown  by  the  interesting  experiments 
recently  published  by  Morgenroth.1  This  author  showed  that  by 
allowing  hydrochloric  acid  to  act  on  a  neutral  mixture  of  cobra 
venom  and  its  antitoxin,  complete  dissociation  could  be  effected, 
so  that  the  entire  amount  of  the  two  substances  could  be  recovered. 
Morgenroth  rightly  regards  this  demonstration  as  an  important 
argument  in  favor  of  the  chemical  theory  of  the  toxin-antitoxin 
reaction,  and  emphasizes  the  fact  that  this  behavior  in  no  way 
contradicts  the  stereochemical  conception  formulated  by  Ehrlich. 

Conditions  apparently  are  such  that  after  the  union  has  become 
firm  only  the  intense  influence  of  powerful  agents,  such  as  hydrochloric 
acid  in  the  case  before  us,  or  ferments  in  the  case  of  glucosides,  are 
able  to  effect  dissociation.  In  contrast  to  this,  we  see  that  the  di- 
lution phenomenon  studied  by  us  is  demonstrable  only  during  the 
stage  of  loose  union,  mere  dilution  being  unable  to  effect  dissociation 
after  the  union  has  become  firm.  It  is  evident,  from  what  has  been 
said,  that  it  is  impossible  to  analyze  these  reactions  according  to  the 
principle  of  the  Guldberg-Waage  law.  Objection  must  also  be  made 
to  the  attempts  to  view  these  relations  from  the  standpoint  of 
colloid  chemistry.  These  attempts  grow  out  of  purely  external 


1  Morgenroth,  Ueber  die  Wiedergewinnung  von  Toxin  aus  seiner  Antitoxin- 
verbindung,  Berliner  klinische  Wochenschrift,  1905,  No.  50. 


DISSOCIATION  IN  THE  TOXIN-ANTITOXIN  COMBINATION.      673 

analogies  which  in  no  way  warrant  abandoning  the  structuro- 
chemical  conception.  The  latter  alone  has  been  able  to  do  justice  to 
the  manifold  phenomena  under  discussion. 

We  have  seen  that  the  increased  toxicity  effected  by  dilution  is 
not  dependent  on  any  special  vital  influences  on  the  part  of  the 
animal  injected.  This  point  is  still  further  confirmed  by  experi- 
ments which  we  made  with  arachnolysin  (the  haBmolytic  principle 
of  the  garden  spider  *),  in  which  we  were  able  to  reproduce  the  same 
conditions  in  test-tube  experiments.2 

The  serum  employed  in  our  experiments  was  obtained  by  im- 
munizing rabbits  against  arachnolysin.  This  poison  is  particularly 
well  suited  for  experiments  of  this  kind  because  it  is  very  resistant 
and  because  the  reaction  between  arachnolysin  and  antilysin  is 
practically  completed  in  an  hour.  During  the  first  hour,  to  be  sure, 
the  course  of  the  reaction  is  a  gradual  one.  The  blood  used  was 
always  1  cc.  of  a  5%  suspension  of  rabbit  blood.  Of  the  arachno- 
lysin 0.2  cc.  (approximately  200  complete  solvent  doses)  were  mixed 
with  varying  amounts  of  antilysin  and  the  mixtures  made  up  to  an 
equal  volume  (8  cc.)  with  physiological  salt  solution.  The  first 
titration  of  the  mixture  was  undertaken  at  the  end  of  an  hour,  and 
a  second  at  the  end  of  24  hours.  The  contents  of  each  tube  was 
always  made  up  to  2  cc.  with  salt  solution.  The  result  of  the 
experiment  is  shown  in  Table  VII. 


1  Sachs,   Zur  Kenntniss  des  Kreuzspinnengiftes.      See    this  volume,  page 
167. 

2  Madsen,  to  be  sure,  mentions  similar  observations  in  the  case  of  saponin 
and  cholesterin.     His  experiments,  however,  do  not  impress  us  as  justifying 
the  analogizing  conclusion  which  he  draws.     Thus  one  sees  that  the  deter- 
minations  of   the   hsemolytic .  power   of   the   saponin   do   not   proceed    quite 
regularly;  the  saponin  by  itself,  in  his  tests,  sometimes  acts  more  powerfully 
in  small  doses  than  in  large.     Then,  too,   in  the  titrations  of  the  saponin- 
cholesterin  mixtures  there  are  zones  of  marked  action  from  which  there  is 
diminished  haemolysis  both  with  larger  and  with  smaller  doses.     Finally,  it 
should  be  noted  that  this  diminution  is  succeeded  upwards  by  a  progressive 
increase  of  haemolysis,   reaching  its    maximum  with  the  largest  dose  of  the 
mixture.     It   is  evident,    therefore,    that   these  experiments  of  Madsen  have 
nothing  to  do  with  the   phenomena  observed  by  him   or  with  those  observed 
by  us   with   the   toxin   of   botulism.     We  are  unable  to   say  what  causes    the 
irregularities  in  the  saponin-cholesterin  tests.     The   mechanism   of    the    action 
of   cholesterin   on    saponin    is     manifestly    entirely    different     from    that    of 
the  toxin-antitoxin  reaction. 


674 


COLLECTED  STUDIES  IN  IMMUNITY. 


TABLE   VII. 

A. 
THE  H^MOLYTIC  POWER  DETERMINED  AT  THE  END  OF  1  HOUR. 


Amount  of 

The  Mixture  was  Composed  of  0.2  cc.  Arachnolysin  +  Antiarachnolysin, 

cc. 

2.4  cc. 

2.0  cc. 

1.6  cc. 

1.2  cc. 

0.8  cc. 

1.0 
0.5 

0 
0 

0 
0 

faint  trace 
trace 

moderate 

<  s 

complete 

0.25 

0 

faint  trace 

moderate 

marked 

i 

0.15 

0 

trace 

complete 

(  ( 

t 

0.1 

0 

1  1 

moderate 

moderate 

t 

0.05 

faint  trace 

slight 

1  1 

" 

' 

0.025 

1  1 

trace 

slight 

slight 

marked 

0.015 

trace 

'  ' 

14 

0.1 

i  ( 

faint  trace 

faint  trace 

1  1 

moderate 

B. 
THE  ELEMOLYTTC  POWER  DETERMINED  AT  THE  END  OF  24  HOURS. 


1.0 

faint  trace 

moderate 

complete 

0.5 

trace 

i  < 

i 

0.25 

marked 

« 

0.15 

" 

t 

0.1 

0 

0 

moderate 

( 

0.05 

1  1 

t 

0.025 

faint  trace 

1  1 

marked 

0.015 

slight 

i 

0.01 

trace 

The  table  shows  that  in  relatively  fresh  mixtures  of  arachno- 
lysin  and  antiarachnolysin  the  dilution  phenomenon  can  be  strikingly 
demonstrated.  With  the  mixtures  which  have  stood  for  24  hours, 
however,  the  power  of  acting  more  strongly  in  smaller  doses  has 
largely  disappeared,  though  even  here  there  is  some  indication  of 
the  curious  phenomenon.  It  fact,  even  after  48  and  72  hours  the 
phenomenon  is  present  to  a  slight  degree.  We  see,  therefore,  that 
the  results  with  arachnolysin  correspond  entirely  with  those  observed 
with  botulism  toxin,  and  the  same  explanation  applies. 

Before  closing  this  paper,  we  must  call  attention  to  a  remarkable 
observation  made  in  the  course  of  these  experiments.  On  resuming, 
this  summer,  the  work  which  we  had  begun  a  year  and  more  before, 
we  found  it  impossible  to  reproduce  the  paradoxical  phenomenon 
with  the  old  sera  left  from  the  original  experiments.  We  therefore 


DISSOCIATION  IN  THE  TOXIN-ANTITOXIN  COMBINATION.      675 

prepared  fresh  antilytic  sera  by  immunizing  rabbits,  and  found  that 
these  at  once  gave  the  paradoxical  results  under  discussion.  Con- 
cerning the  cause  of  this  peculiar  behavior  of  fresh  and  old  anti- 
toxin we  can  only  offer  conjectures.  One  could  assume  that  on 
standing  the  antitoxin  becomes  changed  into  a  form  possessing  greater 
affinity.  It  must  be  admitted  that  the  experiences  had  with  other 
sera,  both  antitoxic  and  bactericidal,  do  not  lend  support  to  this 
assumption,  since  thus  far  we  know  age  merely  to  weaken  the  sera 
but  not  to  increase  the  antitoxic  action.  It  is  more  natural,  there- 
fore, to  assume  that  the  serum  contains  substances  which  act  like 
negative  catalyzers.  Thus,  while  the  positive  catalyzers  already 
mentioned  hasten  the  toxin-antitoxin  reaction,  the  negative  cata- 
lyzers assumed  to  exist  in  the  serum  would  retard  the  tightening 
of  the  union.  One  would  then  say  that  the  fresh  antiarachnolysin 
serum  contained  the  negative  catalyzer,  and  that  this  by  retarding 
the  tightening  of  the  union,  made  possible  the  dissociation  of  the 
two  components  when  the  mixtures  were  diluted.  In  an  old  serum, 
on  the  other  hand,  this  retarding  substance  would  be  absent,  thus 
making  the  toxin-antitoxin  union  firm  in  a  very  short  time.  In 
that  case,  of  course,  the  dilution  phenomenon  could  not  be  demon- 
strated. 

We  believe  that  a  mere  study  of  the  successive  events  in  the 
toxin-antitoxin  neutralization  permits  of  no  direct  conclusions.  We 
have  seen  that  it  is  impossible  to  exclude  certain  factors  which 
markedly  affect  the  course  of  the  reaction;  the  existence  of  positive 
catalyzers  had  necessarily  to  be  assumed,  and  the  influence  of  nega- 
tive catalyzers  was  rendered  probable  by  the  results  of  our  investi- 
gations. It  is  therefore  impossible  by  a  mere  numerical  analysis  of 
the  course  of  the  experiment  to  draw  definite  conclusions  concerning 
the  absolute  combining  affinity  in  the  toxin-antitoxin  reaction. 


XLIX.    THE    PARTIAL-FUNCTIONS   OF  CELLS.* 

By  Prof.  PAUL  EHRLICH. 

THE  history  of  our  knowledge  of  vital  phenomena  and  of  the 
organic  world  can  be  divided  into  two  parts.  For-  a  long  time 
anatomy,  especially  the  anatomy  of  the  human  body,  constituted 
the  beginning  and  the  end  of  scientific  knowledge.  Further  progress 
was  only  made  possible  by  the  invention  of  the  microscope.  Many 
years,  however,  passed  by  before  Schwann  demonstrated  the  cell 
as  the  final  biologic  unit.  It  would  be  like  carrying  wisdom  to 
Athens  to  sketch  for  you  the  immeasurable  progress  which  we  owe 
to  the  introduction  of  the  cell  concept,  the  concept  about  which  the 
entire  modern  science  of  life  turns. 

I  take  it  to  be  generally  accepted  that  everything  which  goes  on 
within  the  body,  assimilation  and  disassimilation,  is  referable,  in 
the  final  analysis,  to  the  cell;  that  the  cells  of  different  organs  are 
differentiated  from  each  other  in  a  specific  manner,  and  that  this 
differentiation  makes  it  possible  for  them  to  fulfill  their  various 
functions. 

The  results  mentioned  were  achieved  principally  by  histological 
examinations  of  dead  and  living  tissues,  though  the  allied  sciences, 
physiology,  toxicology,  and  especially  comparative  anatomy  and 
biology,  made  most  valuable  contributions.  Nevertheless  I  am 
inclined  to  believe  that  the  aid  which  the  microscope  has  given  and 
can  still  give  us  is  approaching  a  limit,  and  that  in  a  deeper  analysis 
of  the  all-important  problem  of  cell  life  the  application  of  optical 
contrivances,  no  matter  how  delicate,  will  fail  us.  The  time  has 
come  for  a  further  study  of  the  minute  chemistry  of  cell  life;  the 
concept  cell  must  be  resolved  into  a  large  number  of  distinct  partial 

1  The  Nobel  Lecture,  delivered  in  Stockholm,  Dec.  11,  1908.  Reprinted 
from  Miinchener  mediz.  Wochenschrift,  No.  5,  1909. 

676 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  677 

functions.  The  activities  of  a  cell,  however,  are  essentially  chemical 
in  nature,  and  since  the  formation  of  chemical  structure  is  beyond 
the  pale  of  visibility,  it  follows  that  we  must  cast  about  for  other 
methods  of  study.  This  is  important  not  only  for  a  real  understand- 
ing of  vital  phenomena,  but  because  it  constitutes  the  basis  of  a. 
truly  rational  use  of  drugs. 

The  first  step  in  this  complicated  domain  was  taken,  as  is  often 
the  case,  quite  indirectly.  Following  Behring's  great  discovery  of 
the  antitoxins,  I  sought  to  gain  a  deeper  insight  into  the  nature  of 
their  action,  and  after  considerable  study  succeeded  in  finding  the 
key  to  the  mystery. 

You  all  know  that  the  power  to  excite  the  production  of  anti- 
bodies is  confined  to  a  distinct  group  of  poisonous  substances,  the 
so-called  toxins.  These  are  products  of  the  metabolism  of  animal 
or  vegetable  cells:  diphtheria  and  tetantus  toxins,  abrin,  ricinv 
snake  venom,  and  many  others.  None  of  these  substances  can  be 
crystallized;  all  seem  to  belong  to  the  class  of  substances  spoken  of 
as  albuminoid.  In  general  the  toxin  is  characterized  by  two  prop- 
erties, first,  its  toxicity,  second,  its  power  to  excite  the  production 
of  a  specific  antitoxin  in  the  animal  body. 

In  my  quantitative  investigations  concerning  this  process  I 
found  that  the  toxins,  especially  solutions  of  diphtheria  toxin, 
underwent  a  peculiar  transformation,  either  spontaneously  on  stand- 
ing, or  through  the  action  of  thermic  or  chemic  influences.  While 
their  toxicity  was  lost  to  a  greater  or  less  extent,  their  power  to 
excite  antibody  production  in  the  animal  body  remained  intact. 
Furthermore,  it  was  found  that  these  transformation  products, 
which  I  term  toxoids  and  which  my  esteemed  friend,  Professor 
Arrhenius,  has  encountered  in  his  numerous  experiments,  these 
toxoids  still  retained  the  power  to  specifically  neutralize  the  anti- 
toxin. In  fact,  in  favorable  cases  it  was  possible  to  demonstrate 
that  the  transformation  of  toxin  into  toxoid  is  quantitative,  i.  e.,  a 
certain  poison  solution  would  neutralize  exactly  the  same  amount  of 
antitoxin  before  as  after  the  transformation  into  toxoid. 

These  facts  permit  of  but  one  explanation,  namely,  that  the 
toxin  possesses  two  groups  having  different  functions.  One  of  these 
which  remains  intact  in  the  "  toxoid  "  and  which  therefore  is  to  be 
regarded  as  the  more  stable,  must  possess  the  property  of  exciting  the 
production  of  antibodies  when  injected  into  an  animal,  and  must 
also  be  able  to  neutralize  the  antibody  both  in  a  test  tube  and  in 


678  COLLECTED  STUDIES  IN  IMMUNITY. 

vivo.  Since,  however,  the  relations  existing  between  toxin  and  its 
antitoxin  are  strictly  specific  (tetanus  antitoxin  neutralizes  only 
tetanus  poison,  diphtheria  serum  only  diphtheria  poison,  snake 
antivennin  only  snake  venom,  etc.,  etc.)  it  is  necessary  to  assume 
that  a  chemical  union  occurs  between  the  two  opposing  substances. 
In  view  of  the  strict  specificity  this  binding  is  best  explained  by 
assuming  the  existence  of  two  groups  having  a  definite  configura- 
tion, of  two  groups  fitting  one  another  like  a  lock  to  a  key,  to  use 
Emil  Fischer's  apt  comparison.  Considering  the  firmness  of  the 
union  on  the  one  hand,  and  the  fact  that  neutralization  takes  place 
even  in  very  high  dilutions  without  the  aid  of  chemical  agents,  we 
must  assume  that  the  binding  is  due  to  a  close  chemical  relationship, 
in  all  probability  analogous  to  a  true  chemical  synthesis. 

Recent  investigations,  in  fact,  have  shown  that  it  is  possible,  by 
chemical  interference,  to  disrupt  the  combination,  to  split  the  toxin- 
antitoxin  union  into  its  components.  Morgenroth,  for  example,  has 
shown  this  with  a  number  of  poisons.  Thus  with  snake  venom  and 
diphtheria  poiosn  he  found  that  the  action  of  hydrochloric  acid 
caused  the  toxin-antitoxin  combination  to  resolve  into  its  original 
components,  just  as  in  pure  chemistry  stable  combinations  such  as 
the  glucosides,  when  acted  on  by  acids,  are  resolved  into  their  two 
components,  sugar  and  the  constituent  aromatic  group.  These 
investigations  showed  that  the  more  stable  group  of  the  toxin 
molecule,  the  group  to  which  I  have  given  the  name  "  haptophore," 
is  able  to  exhibit  marked  chemical  activity  of  specific  character, 
and  it  was  therefore  very  natural  to  assume  that  just  this  group 
effected  the  anchoring  of  the  toxin  to  the  cell.  We  see,  for  example, 
how  many  species  of  bacterial  poisons  take  weeks  before  they  pro- 
duce disturbances,  and  how  they  confine  their  injurious  action  to 
heart,  kidney,  or  nerve.  We  see  animals  ill  of  tetanus  infection 
exhibiting  spasms  and  contractures  for  months.  All  this  compels 
us  to  admit  that  these  phenomena  can  only  be  caused  by  the  anchor- 
ing of  the  poison  by  certain  definite  cell  complexes. 

I  therefore  assumed  that  tetanus  poison,  for  example,  united 
with  certain  definite  chemical  groups  of  the  cell  protoplasm,  partic- 
ularly of  the  protoplasm  of  the  motor  ganglion  cells,  and  I  further 
believed  that  this  chemical  union  was  the  prerequisite  and  the  cause 
of  the  disease.  These  groups  I  termed  "poison  receptors/'  or  simply 
"  receptors."  Wassermann,  through  his  well-known  experiments, 
was  able  to  demonstrate  the  correctness  of  this  view,  by  showing 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  679 

that  normal  brain  substance  is  able  to  neutralize  definite  quantities 
of  tetanus  toxin.  A  number  of  objections  were  made  against  these 
experiments,  but  they  proved  to  carry  no  weight.  I  am  convinced 
that  it  has  been  proven  conclusively  that  the  cells  contain  definite 
chemical  groups  which  bind  the  poison.  And  that  these  groups, 
receptors,  react  with  the  haptophore  portion  of  the  toxin,  is  shown 
by  the  fact  that  it  is  possible  to  immunize  with  toxoids,  in  which, 
of  course,  only  the  haptophore  group  is  present.  We  know  that  this 
haptophore  group  of  the  toxins  must  possess  a  peculiar,  highly 
complex  stereochemical  structure,  and  since  it  reacts  in  exactly  the 
same  manner  both  with  the  antitoxin  and  with  the  cell  receptors,  we 
conclude  that  the  group  contained  in  the  protoplasm,  the  cell  receptor, 
must  be  identical  with  the  "antitoxin"  present  in  solution  in  the 
scrum  of  the  immunized  animals.  In  view  of  the  fact  that  the  cell 
receptor  constitutes  the  preformed  element,  while  the  artificially 
produced  antitoxin  represents  the  result,  i.  e.,  the  secondary  element, 
it  is  most  natural  to  believe  that  the  antitoxin  is  nothing  else  than 
thrust-off  constituents  of  the  cell,  in  fact  surplus  receptors  which 
have  been  thrust  off.  The  explanation  for  this  is  veiy  simple.  It 
is  merely  necessary  to  assume  that  the  various  specific  cell  receptors 
which  bind,  for  example,  snake  vemon,  diphtheria  poison,  tetanus 
poison,  botulism  poison,  etc.,  are  not  intended  to  serve  as  poison 
catchers  for  poisons  with  which  the  animal  perhaps  never  comes  into 
contact  under  ordinary  conditions,  but  that  they  are  really  designed 
to  chemically  bind  normal  metabolic  products,  i.  e.,  that  they  are 
intended  primarily  to  effect  assimilation.  These  receptors  are  there-, 
fore  to  be  thought  of  as  side  chains  of  the  protoplasm  possessing  the 
power  of  assimilation.  When  laid  hold  of  by  a  toxin  molecule,  the 
particular  normal  function  of  this  group  is  lost,  put  out  of  action. 
Thereupon,  following  the  principle  discovered  by  Weigert,  the  pro- 
toplasm not  only  renairs  the  injury,  but  even  over-compensates  the 
defect,  i.  e.,  there  is  superregeneration.  Finally,  with  the  accumu- 
lation and  repetition  of  the  injections,  so  many  of  these  regenerated 
groups  are  formed  in  the  body  of  the  cell  that  they  hinder,  as  it 
were,  the  normal  cell  functions,  whereupon  the  cell  rids  itself  of  the 
burden  by  thrusting  the  groups  off  into  the  blood. 

The  most  striking  thing  about  this  process  is  the  enormous 
difference  between  the  amount  of  poison  injected  and  the  antitoxin 
produced.  Some  idea  of  this  disproportion  can  be  gained  from  the 
statement  made  by  Knorr  that  one  part  of  toxin  produces  a  quantity 


680  COLLECTED  STUDIES  IN  IMMUNITY. 

of  antitoxin  sufficient  to  neutralize  one  million  times  the  quantity  of 
toxin  injected. 

There  are  those,  to  be  sure,  who  believe  the  process  is  much 
simpler  than  this.  Straub,  for  example,  thinks  it  is  essentially 
analogous  to  simple  detoxicating  phenomena  occurring  in  the  body, 
comparing  it,  for  example,  with  the  formation  of  an  ethereal  sul- 
phuric acid  from  injected  phenol.  The  only  difference,  Straub 
believes,  is  that  phenol  sulphuric  acid  is  stable  in  the  organism,  while 
the  toxin-antitoxin  combination  is  unstable,  being  partially  destroyed 
in  the  organism.  This  destruction,  however,  affects  only  one  com- 
ponent, the  injected  toxin,  the  other,  the  reaction  product  of  the 
organism  (being  related  to  the  organism  and  therefore  not  a  foreign 
biological  substance)  escapes  elimination  and  remains  in  the  blood 
and  body  fluids.  By  systematically  repeating  the  poisoning  it  is 
thus  possible  to  increase  the  protective  power  of  the  blood,  so  that 
when  this  blood  is  injected  into  other  animals  the  protective  power 
is  transformed,  and  the  injected  animals  become  resistant  to  the 
toxic  infection. 

This  is  Straub's  idea.  With  so  simple  an  explanation,  one  will 
wonder  why  this  question  has  engaged  the  attention  of  so  many 
investigators  in  immunity  these  many  years.  As  a  matter  of  fact, 
however,  it  seems  entirely  to  have  escaped  the  author  that  according 
to  his  theory  a  certain  quantity  of  toxin  can  only  produce  an  equiv- 
alent amount  of  antitoxin.  Fortunately,  however,  in  immuniza- 
tion this  is  not  the  case.  It  can  be  shown,  as  has  already  been  said, 
that  one  part  of  toxin  can  produce  an  amount  of  antitoxin  a  million 
times  more  than  the  equivalent.  This  alone  is  enough  to  show 
how  untenable  Straub's  conception  is. 

Of  far  greater  importance  is  the  fact  that  the  demonstration  of 
this  hyperregeneration  proves  the  preformation  and  the  chemical 
individuality  of  the  corresponding  toxin  receptors.  That  which  the 
cell  constantly  produces  and  which  can  be  given  off  to  the  blood  after 
the  manner  of  a  secretion  must  have  a  chemical  "individuality." 
This  constitutes  the  first  step  toward  resolving  the  cell  concept  into 
a  large  number  of  separate  individual  functions.  From  the  begin- 
ning I  had  assumed  that  the  toxin  represented  nothing  more  than  an 
assimilable  food  stuff  to  which  in  addition,  by  chance  as  it  were,  was 
attached  a  side  group,  very  labile  in  character,  which  really  exerted 
the  toxic  action. 

This  view  was  very  quickly  confirmed  in  a  number  of  ways. 


THE   PARTIAL-FUNCTIONS   OF   CELLS.  681 

The  actual  independence  of  haptophore  and  toxophore  groups  was 
conclusively  demonstrated  by  the  discovery  of  substances  which 
had  the  power  to  excite  the  production  of  antibodies,  and  which, 
therefore,  were  antigens,  without  possessing  any  toxic  action.  I 
may  remind  you  of  the  precipitins  first  observed  by  Kraus,  Tschis- 
tovitsch  and  Bordet.  These  authors  showed  that  albuminous  bodies 
derived  from  either  animal  or  vegetable  organisms  were  able  to 
excite  the  production  of  specifically  reacting  antibodies,  and  this 
whether  they  possessed  toxic  properties  or  not.  The  demonstration 
of  their  antigen  nature  was  thus  extended  to  true  food- stuffs,  a  result 
to  be  expected  on  the  basis  of  my  theory.  Moreover,  even  among 
the  poisons  found  in  nature,  some  have  been  encountered  in  which 
the  independence  of  the  haptophore  and  of  the  toxophore  apparatus 
is  at  once  recognized.  I  refer  to  cytotoxins  which  are  found  normally 
in  the  blood  serum  of  certain  higher  animals,  or  which  can  be  artifi- 
cially produced  by  immunization  with  any  particular  species  of  cell. 
These  cytotoxins  differ  from  all  other  poisons  known  to  us  by  the 
extraordinary  specificity  of  their  action  by  a  degree  of  monotropism 
possessed,  so  far  as  we  know,  only  by  the  poisons  derived  from  the 
living  animal  body.  Owing  to  their  complex  constitution  it  is  easy 
to  differentiate  the  haptophore  and  the  toxophore  apparatus,  and 
to  show  that  ihe  function  of  the  distributive  component,  the  ambo- 
ceptor,  is  to  concentrate  the  really  active  substance  on  the  affected 
cell.  This  is  effected  by  an  increase  in  the  affinity  of  the  amboceptor 
after  union  with  the  cell  has  taken  place.  The  fact  that  ani- 
mal cells  act  as  antigens  without  possessing  any  toxic  action,  and  the 
fact  that  it  is  possible  to  immunize  with  dissolved  albuminous  sub- 
stances, demonstrates  that  only  the  haptophore  group  is  responsible 
for  the  formation  of  antibodies. 

The  recognition  and  the  careful  analysis  of  the  specific  relations 
existing  between  the  haptophore  groups  of  antibodies  and  of  recep- 
tors, has  proven  of  the  highest  theoretical  and  practical  importance 
in  serum  diagnosis.  To  cite  only  a  few  examples,  let  me  call  your 
attention  to  the  determination  of  the  agglutinating  titer  in  its 
application  to  the  Widal  reaction  in  typhoid  fever,  to  the  method 
of  differentiating  albumins  introduced  by  Wassermann  and  Uhlen- 
huth,  and  its  significance  in  the  forensic  diagnosis  of  blood,  to  the 
measurement  of  the  opsonic  index  introduced  by  Wright,  and  to 
numerous  applications  which  haVe  been  made  of  the  method  of 
complement  binding,  a  method  whose  scientific  basis  also  rests 


682    *  COLLECTED  STUDIES  IN  IMMUNITY. 

on  the  principle  of  anchoring  the  antibody  to  the  haptophore 
group. 

Without  going  further  into  this  subject,  I  wish  merely  to  em- 
phasize the  fact  that  there  are  a  number  of  foodstuffs,  mostly  probably 
albuminous  in  character,  which  find  specific  receptors  on  the  cells, 
and  that  we  are  thus  enabled  by  means  of  immunization  to  draw 
these  receptors  into  the  blood.  Here  they  present  themselves  in 
various  forms  as  agglutinins,  precipitins,  amboceptors,  and  opsonins, 
and  as  antitoxins  and  antiferments.  By  causing  them  to  accumulate 
in  the  blood  we  can  subject  these  substances  to  minute  analysis, 
a  procedure  entirely  out  of  the  question  so  long  as  they  remained 
part  of  the  cell.  The  extent  to  which  the  analysis  of  these  reactions 
can  be  pursued  is  well  illustrated  by  the  study  of  the  toxin-antitoxin 
combination  and  by  the  recognition  of  the  complex  character  of  the 
amboceptor  action. 

This,  of  course,  does  not  by  itself  solve  the  mystery  of  life.  Com- 
paring the  latter  to  the  complex  structure  of  a  mechanical  apparatus, 
we  might  say  that  we  are  at  least  able  to  take  out  some  of  the  wheels 
and  study  them  minutely.  This  is  certainly  a  great  advance  over 
the  former  method — to  smash  the  entire  apparatus  and  then  hope 
to  learn  something  from  the  mass  of  fragments. 

I  term  all  the  receptors  which  are  enabled  and  designed  to  assimi- 
late foodstuffs  for  the  cell  "nutri-receptors."  I  consider  that  these 
nutri-receptors  constitute  the  source  of  the  antibodies  mentioned 
above.  From  a  pluralistic  standpoint  it  is,  of  course,  necessary  to 
assume  that  there  are  a  large  number  of  nutri-receptors  of  various 
kinds.  In  view  of  the  complexity  of  the  organism,  and  of  the 
multiplicity  and  specificity  of  the  cell  functions,  a  standpoint  other 
than  this  appears  out  of  the  question.  In  immunizing  we  can  dis- 
tinguish three  classes  of  nutri-receptors,  namely: 

1.  Those  which  do  not  pass  into  the  blood  in  the  form  of  anti- 
bodies.    We  may  assume  that  this  is  the  case  with  nutri-receptors 
serving  the  very  simplest  functions,  as,  for  example,  the  absorption 
of  simple  fats  and  sugars. 

2.  Those  which  pass  into  the  blood  in  the  manner  described 
above,  forming  characteristic  antibodies.     The  production  of  these 
corresponds  to  a  superregeneration. 

3.  The  third  form  contrasts  with  the  preceding,  in  that  instead 
of  a  regeneration,  there  is  a  disappearance  of  receptors.     Experi- 
mental evidence  of  the  occurrence  of  this  form,  to  be  sure,  has  thus 


THE  PARTIAL-FUNCTIONS   OF   CELLS.  683 

far  been  very  meagre.  The  one  example  which  may  be  familiar  to 
the  reader  is  the  fact  demonstrated  by  H.  Kossel  that  on  long- 
continued  immunization  of  rabbits  with  the  hsemotoxic  eel  serum, 
the  blood  cells  finally  became  insusceptible  to  this  serum,  as  though 
they  had  lost  their  specific  receptors. 

Recently,  aided  by  my  colleagues,  Dr.  Rohl  and  Miss  Gulbransen, 
I  succeeded  in  gaining  an  insight  into  the  nature  of  the  disappearance 
of  receptors.  While  the  work  will  be  made  the  subject  of  a  special 
paper,  I  may  here  say  that  our  experiments  were  made  on  trypano- 
somes.  Working  in  my  laboratory,  Franke,  after  infecting  a  monkey 
with  a  particular  species  of  trypanosome,  had  cured  the  disease  by 
means  of  chemo-therapeutic  agents,  and  had  tested  the  immunity 
of  the  animal  by  again  infecting  it  with  the  original  strain.  Con- 
trary to  expectations,  it  was  found  that  the  monkey  was  not  immune, 
so  that  after  a  very  prolonged  incubation,  the  disease  reappeared. 
If  mice  were  inoculated  with  blood  from  the  diseased  animal,  i.  e., 
with  blood  containing  trypanosornes,  they  became  infected  and 
died.  Curiously,  however,  if  the  trypanosomes  were  first  removed 
from  this  monkey  blood,  it  was  found  that  the  serum  was  able  to 
kill  the  original  strain  of  trypanosomes.  This  showed  that  the  try- 
panosomes had  undergone  some  change  in  the  body  of  the  monkey, 
and  that  the  variety  thus  produced  differed  from  the  original  strain 
in  its  behavior  toward  the  serum;  it  had  become  serum-fast. 
Similar  observations  were  made  at  the  same  time  by  Kleine,  and 
recently  also  by  Mesnil. 

We  found  that  when  animals  which  had  been  infected  with  a 
particular  strain  of  trypanosome  were  treated  with  less  than  the 
complete  sterilizing  dose  of  suitable  substance  (arsanil,  arsazetin, 
arsenophenylglycin)  the  trypanosornes  disappear  from  the  blood  for 
a  time.  It  can  easily  be  shown  that  in  this  case  also  antibody  has 
been  produced.  The  few  parasites  which  escape  destruction  lie 
dormant  in  the  body  for  a  time  and  gradually  adapt  themselves  to 
the  antibodies  present  in  the  serum.  Then  they  again  pass  into  the 
blood,  where  they  rapidly  multiply  and  bring  about  the  death  of  the 
animal.  We  inoculated  the  trypanosomes  so  obtained  into  two 
series  of  mice.  One  series  consisted  of  mice  which  had  been  infected 
with  the  original  strain  and  then  cured  with  suitable  doses.  These 
animals,  therefore,  possessed  specific  antibodies.  The  other  series 
consisted  of  normal  mice.  Infection  resulted  equally  rapidly  hi 
both  series.  This  shows  that  the  parasites  of  the  strain  producing 


684  COLLECTED  STUDIES  IN  IMMUNITY. 

the  relapse  have  undergone  a  biological  alteration,  in  that  they  have 
become  serum-fast.1  The  alteration  in  these  parasites  is  not  super- 
ficial in  character.  On  the  contrary  it  may  persist  for  many  months 
and  through  repeated  passage  through  normal  animals.  The  re- 
lapse strain  maintains  its  resistance  to  the  antibodies  produced  by 
the  original  strain,  and  can  thus  be  positively  identified. 

It  was  necessary  to  attempt  to  gain  an  insight  into  the 
nature  of  this  alteration.  After  varying  the  experiments  in  many 
ways  we  reached  the  following  conclusion:  The  original  strain  is 
plentifully  supplied  with  a  certain  uniform  type  of  nutri-receptor, 
which  we  may  term  group  A.  If  the  parasites  are  now  killed  and 
dissolved  in  the  mouse's  body,  group  A  acts  as  antigen  and  gives 
rise  to  antibodies  having  definite  relationship  to  group  A.  When 
living  parasites  are  brought  into  contact  with  this  antibody,  either 
in  vitro  or  in  vivo,  the  antibody  is  anchored  by  the  parasites.  As 
a  result  of  this  occupation  of  its  receptors,  the  parasjtes  undergo  the 
biological  alteration  which  leads  to  the  relapse  strain.  The  altera- 
tion consists  in  the  disappearance  of  the  original  group  A,  and  its 
replacement  by  a  new  group,  B.  The  following  experiment  shows 
that  the  relapse  strain  contains  a  new  group.  Two  mice  are  infected 
with  the  relapse  strain,  which  possesses  group  B,  and  are  then  com- 
pletely healed.  On  infecting  one  mouse  with  the  original  strain, 
the  other  with  the  relapse  strain,  it  will  be  found  that  infection  with 
the  original  strain,  carrier  of  group  A,  is  successful,  while  reinfection 
with  the  relapse  strain  is  at  first  unsuccessful.  This  shows  that  the 
original  strain  and  the  relapse  strain  are  not  identical,  that  they 
must  be  carriers  of  two  different  functional  groups.  We  are  dealing, 
therefore,  with  a  typical  case  of  disappearance  of  receptors  following 
immunization,  and  accompanied  by  the  formation  of  an  entirely  new 
variety  of  receptor. 

It  is  probably  of  little  consequence  whether  this  alteration  is 
regarded  as  a  mutation  or  a  variation.  The  important  thing  is 
that  it  can  be  artificially  produced  at  will,  and  that  it  is  hereditary. 
In  view  of  the  great  interest  attaching  to  this  problem  in  biology  and 
embryology,  we  have  attempted  a  further  analysis  of  the  phenomenon. 

1  Exactly  the  same  strain  can  be  produced  in  much  simpler  fashion,  by 
infecting  mice  with  the  original  strain,  and  healing  the  animals  on  the  second 
day  with  a  full  healing  dose.  After  two  or  three  days  they  are  then  again 
infected  with  the  same  strain.  After  a  time  parasites  will  appear  in  the  blood> 
and  these  will  be  found  to  correspond  entirely  to  those  of  a  relapse  strain. 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  685 

To  begin,  it  was  necessary  to  determine  in  what  manner  the 
trypanosome  antibodies  affected  the  parasites.  Corresponding  to 
our  previous  knowledge  of  immunity  we  could  assume  that  these 
antibodies  exert  a  direct  poisondus  action,  i.  e.,  that  they  therefore 
probably  contained  toxophore  or  tiypanolytic  groups,  so  that  the 
anchoring  of  the  antibody  by  the  parasite  is  followed  by  an  injury 
or  even  the  destruction  of  the  latter.  This,  however,  is  not  the  case. 
In  contrast  to  the  ordinary  strains  of  trypanosomes,  which  possess 
only  a  uniform  group,  A,  B,  or  C,  and  which  may  therefore  be  termed 
"Unios,"  one  meets  with  other  strains  which  possess  two  groups  in 
their  protoplasm,  A  and  B,  and  which  may  therefore  be  termed 
"Binios."  If  such  a  binio  "A-B"  is  acted  on  by  the  isolated 
antibody  A  or  B,  growth  will  not  be  injured  in  the  least.  Not  until 
both  antibodies  act  at  once  does  this  occur.  From  this  it  follows 
that  the  presence  of  the  antibodies  does  not  produce  a  direct  toxic 
effect  on  the  parasites.  To  us  it  seems  that  this  three-fold  experi- 
ment demonstrates  that  the  antibody  acts  merely  by  blocking  the 
food  supply  by  occupying  the  corresponding  receptors.  It  thus 
comes  to  pass  that  when  in  the  binio  A-B  the  group  A  is  occupied- 
by  an  antibody,  the  parasite  can  continue  to  vegetate  by  means  of 
the  group  B.  From  this  it  also  follows  that  groups  A  and  B  are 
essentially  nutri-receptors. 

If  the  amount  of  antibody  is  very  large,  the  parasite  finds  it 
impossible  to  obtain  nourishment,  and  consequently  dies  off.  This 
can  easily  be  demonstrated  by  mixing  the  parasite  in  a  test  tube  with 
varying  amounts  of  antiserum;  the  parasite  is  killed  in  the  high 
concentrations  which  completely  shut  off  the  food  supply,  while  in 
the  weaker  concentrations,  which  permit  a  vita  minima,  the  parasites 
undergo  the  alteration  already  discussed,  and  give  rise  to  a  relapse 
strain.  This  mutation  is  therefore  referable  entirely  to  a  hunger 
of  the  protoplasm,  and  under  this  .influence  the  trypanosome  de- 
velops new  potentialities.  I  have  given  the  name  "atrepsins"  to 
antibodies  of  the  type  just  discussed,  i.  e.,  those  whose  action  is 
purely  antinutritive,  and  I  believe  that  they  play  an  important  role 
not  only  with  bacteria  but  in  biology  in  general. 

In  view  of  the  fact  that  the  presence  of  antibodies  demonstrates 
the  existence  of  definite  chemical  groupings,  most  of  the  workers  in 
immunity  will  have  no  difficulty  in  accepting  the  idea  that  there  are 
definite  chemical  groups  in  the  cell  designed  for  the  taking  up  of 
nutritive  material.  A  much  more  difficult  question  is  as  to  the. 


686  COLLECTED  STUDIES  IN  IMMUNITY. 

existence  of  analogous  groups  for  the  assimilation  of  less  complex 
substances.  So  far  as  the  simplest  additional  function  is  concerned, 
namely,  the  absorption  of  oxygen,  I  believe  this  question  is  already 
partly  answered.  It  is  well  established  that  in  the  haemoglobin 
molecule  it  is  exclusively  the  organically  bound  iron  residue  which 
effects  the  loose  union  with  the  oxygen  on  the  one  hand,  and  the 
carbon  dioxide  and  hydrocyanic  acid  on  the  other.  It  will  therefore 
be  necessary  to  assume  that  the  red  blood  corpuscles  contain 
definite  groupings  which  possess  a  maximum  affinity  for  iron  and 
with  that  form  a  complex  combination  having  the  characteristic 
functional  properties.  The  protoplasm  of  the  red  blood  corpuscles 
would  thus  be  characterized  by  a  plentiful  supply  of  "  f erro-recep- 
tors,"  the  completing  of  which  receptors  with  iron  leads  to  the 
finished  hsemoglobin  molecule.  Similarly  we  shall  have  to  assume 
the  existence  of  "  cupri-receptors "  in  the  blue  respiratory  pigment 
of  crabs,  and  perhaps  of  "  mangano-receptors "  in  other  animals. 
The  localization  of  iodine  in  certain  glands,  especially  in  the  thyroid 
gland,  and  also  the  fact  that  the  iodine  is  associated  with  certain 
aromatic  side  chains,  will  also  be  interpreted  according  to  this 
conception. 

The  question  as  to  whether  the  cell  contains  preformed  chemo- 
receptors  for  the  great  host  of  true  therapeutic  substances  is  one  of 
great  difficulty.  This  leads  us  into  the  important  domain  governing 
the  relation  between  chemical  constitution  and  pharmacological 
action,  which  in  turn  constitutes  the  basis  for  the  rational  develop- 
ment of  therapeutics.  Not  until  we  have  really  learned  the  site  of 
attack  of  the  parasites,  when  we  have"  come  to  know  what  I  term 
the  therapeutic  biology  of  the  parasites,  will  we  wage  successful 
warfare  against  the  producers  of  infection. 

For  this  reason  I  have  begun  studying  the  existence  of 
particular  chemo-receptors  on  unicellular  organisms,  because  here 
the  conditions  are  much  more  favorable  for  gaining  a  clear  insight 
than  is  the  case  in  the  extremely  complex  mechanism  of  the  higher 
organisms.  The  problem  I  undertook  to  solve  was  this:  Do  trypan- 
osomes  possess,  in  their  protoplasm,  definite  groupings  which  bring 
about  the  anchoring  of  certain  particular  chemical  substances? 

If  any  particular  substance  possesses  the  power  to  kill  trypano- 
somes  or  other  parasites  in  a  test  tube  or  in  the  animal  body,  it  is 
obvious  that  this  can  only  be  due  to  the  fact  that  the  substance  is 
taken  up  by  the  parasites.  This  bald  fact,  however,  does  not  by 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  687 

itself  give  us  an  insight  into  the  way  in  which  this  is  brought  about. 
A  large  number  of  different  explanations  can  be  brought  forward. 
Xot  until  we  can  prove  that  we  are  dealing  with  a  function  which  is 
capable  of  being  altered  and  varied  in  a  specific  manner  is  it  possible 
to  regard  the  existence  of  preformed  groups  as  demonstrated. 

Unfortunately  it  seems  to  be  impossible  to  utilize  the  method 
employed  in  demonstrating  the  preformed  existence  of  nutri-recep- 
tors,  namely,  by  causing  the  liberated  receptors  to  be  thrust  off 
into  the  blood.  The  chemo-receptors  appear  to  be  much  more 
simply  constituted,  and  remain  attached  to  the  cell,  so  that  no 
thrusting-off  occurs. 

By  indirect  means,  however,  we  succeeded  in  getting  light  on 
this  phase  of  the  subject.  With  the  aid  of  my  esteemed  collab- 
orators, Franke,  Browning  and  Rohl,  I  was  able  to  show  that  it  is 
possible,  by  systematic  treatment,  to  produce  strains  of  trypano- 
somes  possessing  immunity  against  the  three  trypanocidal  poisons 
now  known  to  us.  These  poisons,  it  will  be  remembered  are 
(a)  substances  of  the  arsenic  group,  (b)  fuchsin,  and  (c)  the  acid 
azo  dye  known  as  trypanred  belonging  to  the  benzoburpurin  series. 
The  immune  strains  are  marked  by  twro  characteristics: 

1.  A  stability  of  the  acquired  character.     This  is  very  great. 
Thus  our  arsenic  strain,  after  having  been  passed  some  380  times 
through  mice  in  the  course  of  two  and  one-half  years,  still  possesses 
the  same  drug  immunity  as  the  original  strain. 

2.  An  essential  feature  of  the  immunity  to  drugs  is  the  strict 
specificity.     This  manifests  itself  by  the  fact  that  the  immunity  is 
related  not  against  a  certain  definite  elementary  combination,  but 
against  the  entire  chemical  group  of  which  this  combination  is  a 
part.     Thus  the  strain  made  immune  against  fuchsin  is  resistant 
not  only  to  that  substance  but  also  against  a  large  number  of  related 
triphenylmethane  dyes,  e.  g.,  malachite  green,  ethyl  green,  hexae- 
thyl  violet.     In  contrast  to  this,  however,  the  strain  has  remained 
susceptible  to  the  action  of  the  two  other  types,  i.  e.,  against  try- 
panred and  against  an  arsenical.     A  corresponding  specific  resistance 
is  exhibited  by  the  strain  made  fast  against  trypanred  and  by  the 
one   made   fast   against   arsenic   preparations.     That   we   are   here 
dealing  with  three  different  functions  is  further  shown  by  the  fact 
that  by  successive  treatment  of  a  given  strain  with  the  three  sub- 
stances mentioned  above  we  can  produce  a  strain  which  is  resistant 
against  all  three  classes  of  substances,  i.  e.,  one  which  is  triple  fast. 


688  COLLECTED  STUDIES  IN  IMMUNITY. 

Provided  that  the  resistance  thus  produced  is  of  maximum  intensity, 
such  a  strain  is  extremely  useful  in  identifying  new  types  of  try- 
panocidal agents.  If,  for  example,  a  new  substance  is  encountered 
which  is  able  to  kill  ordinary  trypanosomes,  we  have  merely  to 
test  its  action  on  this  triple-fast  strain  in  order  to  determine  whether 
the  substance  really  represents  a  new  type  of  trypanocidal  agent. 
If  it  does  not,  we  shall  find  that  treatment  with  this  substance  does 
not  cause  the  parasites  to  disappear;  on  the  contrary  they  multiply. 
If  they  disappear,  however,  we  can  conclude  that  the  substance  does 
not  correspond  to  any  of  the  three  types  mentioned,  but  represents 
a  new  type  of  trypanocidal  agent.  The  triple-fast  strain  thus  acts 
as  a  kind  of  cribrum  therapeuticum,  by  the  aid  of  which  it  is  possible 
to  recognize  substances  belonging  together  and  to  separate  unrelated 
substances. 

It  was  now  necessary  to  determine  in  what  manner  this  specific 
drug  resistance  is  brought  about,  and  for  this  purpose  I  undertook 
.a  series  of  experiments  with  the  atoxyl  strain.  In  order  to  gain  a  clear 
insight  into  the  question  it  seemed  advisable  to  study  the  behavior 
of  the  arsenic-fast  strains,  also  in  a  test  tube,  away  from  all  disturb- 
ances and  complications  of  the  animal  organism.  This  method 
very  soon  encountered  a  great  obstacle,  for  it  was  found  that  the 
drug  mostly  used  therapeutically,  namely,  atoxyl  (paramidophenyl- 
arsinic  acid),  does  not  exert  the  least  destructive  action  on  try- 
panosomes in  test-tube  experiments.  Even  solutions  containing 
several  per  cent,  of  the  substance  proved  insufficient  for  this  pur- 
pose. This  phenomenon  was  all  the  more  remarkable  because  in 
the  human  body,  according  to  Koch,  injections  of  0.5  g.  atoxyl 
suffice  to  cause  the  disappearance  of  the  parasites  within  a  few  hours. 
In  this  case,  therefore,  destruction  is  effected  in  a  concentration 
of  1  to  120,000. 

We  are  here  dealing  with  a  phenomenon  which  is  usually  spoken 
of  as  "indirect  action."  It  was  not  difficult  for  rne  to  discover  the 
reason  for  this  peculiar  behavior,  as  I  had  for  years  busied  myself 
with  reducing  power  of  the  animal  organism.  We  know  that  in 
the  body  arsenic  acid  is  transformed  into  arsenious  acid;  that 
cacodylic  acid  is  reduced  to  the  ill-smelling  cacodyl.  It  was  natural, 
therefore,  to  think  first  of  reductions.  In  atoxyl,  paramidophenyl- 
arsinic  acid,  the  arsenic  is  pentavalent,  whereas  in  the  two  products 
obtained  from  atoxyl  by  reduction  the  arsenic  is  trivalent.  In 
this  way  we  obtained  two  different  products:  1.  The  monomo- 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  689 

lecular  p-aminophenylarsenoxid  and  2.  The    further    product,  ob- 
tained from  the  latter  by  reduction,  the  yellow  diamidoarsenobenzol. 

In  contrast  to  atoxyl,  these  substances  exhibited  marked  try- 
panocidal  properties  not  only  in  the  animal  body  but  also  in  the 
test  tube.  Thus  a  solution  of  the  arsenoxid  combination  of  a 
strength  of  1  to  1,000,000  killed  the  tryponasomes  in  an  hour.  The 
closely  related  p-oxyphenylarsenoxid  was  still  stronger  killing  in 
1  to  10,000,000. 

This  proved  that  the  pentavalent  arsenic  residue  possesses  no 
trypanocidal  properties  whatever;  these  are  bound  exclusively  to 
the  trivalent,  unsaturated  form. 

As  long  as  sixty  years  ago,  Bunsen,  with  extraordinary  insight, 
pointed  out  that  cacodyl,  the  reduction  product,  is  extremely 
poisonous,  while  cacodylic  acid  is  almost  non-toxic.  This  gave  him 
the  clue  to  the  chemical  character  of  the  cacodyl  combination.  In 
striking  agreement  with  this  is  the  fact  that  the  unsaturated  carbon 
oxid,  for  example,  and  a  number  of  other  unsaturated  combinations 
are  so  much  more  toxic  that  the  corresponding  saturated  combina- 
tions. We  shall,  therefore,  have  to  assume  that  the  arseno-receptor 
of  the  cells  is  able  to  take  up  only  the  unsaiurated  arsenic  residue, 
i.e.,  the  group  possessing  the  greater  combining  affinity. 

With  the  aid  of  such  reduced  combinations  it  was  simply  a  matter 
to  test  the  atoxyl  strain  in  test-tube  experiments.  These  showed 
that  the  organisms  could  be  killed  with  a  suitable  concentration  of 
the  chemical  substances,  and  that  we  were  not  dealing  with  a  loss 
of  receptors  as  in  the  case  of  the  relapse  strain.  A  comparison, 
however,  of  the  lethal  dose  with  the  dose  sufficient  to  kill  the  ordinary 
strain,  showed  that  the  resistant  strain  required  a  much  higher 
concentration.  Amounts  which  effected  immediate  destruction  of 
the  ordinary  strain  did  not  in  the  least  affect  the  vitality  of  the 
resistant  parasites,  even  after  one  hour. 

These  test  tube  experiments  seemed  to  indicate  that  the  arseno- 
receptor,  while  still  preserved  in  the  atoxyl-f ast  strain,  had  undergone 
some  modification  so  that  its  affinity  had  become  lessened.  This 
manifests  itself  by  the  fact  that  it  required  much  stronger  solutions 
to  produce  the  poison  concentration  necessary  to  effect  destruction 
of  the  parasites;  the  normal  arseno-receptor  of  the  original  strain, 
by  virtue  of  its  higher  affinity,  takes  up  the  same  amount  even  from 
more  dilute  solutions. 

We  have  succeeded  in  clearly  demonstrating  by  biological  methods 


690  COLLECTED  STUDIES  IN  IMMUNITY. 

that  the  arseno-receptor  actually  represents  a  distinct  function 
whose  affinity  can  be  systematically  decreased  step  by  step  by  immuni- 
zation. Thus  far  we  have  obtained  three  degrees  of  affinity.  Grade 
I  was  produced  by  subjecting  the  parasites  systematically  to  the 
action  of  p-amidophenylarsinic  acid  and  its  acetyl  combination.  We 
carried  out  this  treatment  ad  maximum  for  years,  until  finally  no 
further  increase  in  resistance  was  produced.  The  resistant  strain 
thus  obtained  proved  to  be  resistant  at  the  same  time  to  a  number 
of  other  arsenicals,  among  them  particularly,  the  p-oxycombination, 
the  combination  with  urea,  and  with  benzyliden,  and  a  number  of 
acid  derivatives. 

In  practical  therapeutics  in  man  and  animals,  it  is,  of  course, 
possible  that  arsenic-fast  strains  develop;  and  these,  naturally,  will 
absolutely  hinder  therapeutic  success.  In  animal  experiments  this 
is  a  common  occurrence.  In  view  of  this  it  is  important  to  discover 
substances  able  still  to  attack  these  resistant  strains,  substances  able 
to  combine  with  their  receptors.  After  long  search  we  found  alto- 
gether three  combinations,  of  which  the  most  important  is  arseno- 
phenylglycin.  With  the  aid  of  this  combination  it  is  possible  to  heal 
infections  produced  by  the  arsenic-fast  strain  I,  which  was  described 
above.  This  can  only  be  explained  by  assuming  that  the  arsenophenyl- 
glycin  lays  hold  on  what  is  left  of  the  arseno-receptor,  somewhat  as  a 
stump  is  grasped  by  a  pair  of  pliers.  The  anchoring  of  this  substance, 
however,  furnishes  a  possibility  for  still  further  increasing  the  arsenic- 
resistance  of  the  strain.  After  considerable  effort  we  succeeded  in 
producing,  out  of  arsenic  strain  I,  a  more  resistant  strain,  arsenic 
strain  II,  which  was  entirely  unaffected  by  arsenophenylglycin. 

Plimmer  has  recently  called  attention  to  tartar  emetic  as  a  sub- 
stance which  kills  trypanosomes,  even  in  high  dilutions.  Tartar 
emetic  is  the  salt  of  an  antimony  combination,  and  antimony,  it  is 
well  known,  is  closely  related  to  arsenic.  On  testing  arsenic  strain  II 
with  tartar  emetic,  we  found  that  the  parasites  were  destroyed  by 
the  tartar  emetic.  By  treating  arsenic  strain  II  with  arsenious 
acid  we  were  able  to  produce  a  still  further  increase  in  resistance, 
so  that  arsenic  strain  III  was  resistant  even  against  tartar  emetic. 
I  want  to  call  particular  attention  to  the  fact  that  this  arsenic  strain 
III,  produced  only  under  the  influence  of  arsenious  acid,  was  re- 
sistant to  tartar  emetic  but  not  against  arsenious  add.  This  can  only 
be  explained  by  assuming  that  of  all  conceivable  arsenicals,  arsenious 
acid  is  the  one  possessing  the  greatest  affinity  to  the  arsenic  receptor, 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  691 

and  that  only  by  the  greatest  effort,  if  at  all,  will  it  be  possible  to 
produce  a  strain  (which  would  be  arsenic  strain  IV)  lesistant  alsa 
against  arsenious  acid. 

I  can  adduce  many  other  interesting  facts  to  support  my  view 
that  under  the  influence  and  attack  of  selected  combinations,  there 
is  a  successive  decrease  in  the  affinity  of  the  receptor  for  that  com- 
bination. Thus,  we  have  found  that  we  can  at  once  employ  one,  of 
the  stronger  agents  producing  resistant  strains,  using,  for  example, 
arsenophenylglycin.  Corresponding  entirely  to  our  expectations, 
the  strain  thus  produced  proved  resistant  also  against  the  less 
powerful  substances,  such  as  atoxyl,  arsacetin,  etc.  A  pan-resistant 
strain  would,  therefore,  be  obtained  if  from  the  outset  we  employed 
the  most  powerful  agents,  namely,  tartar  emetic  and  arsenious  acid. 
Unfortunately,  it  appears  from  our  work  that  it  is  impossible,  at 
least  in  small  laboratory  animals,  to  directly  use  these  substances  for 
this  purpose:  it  is  necessary  to  proceed  indirectly,  by  treating  the 
organisms  first  with  phenylarsinic  acid  derivatives. 

The  loss  of  affinity  is,  of  course,  a  chemical  phenomenon,  and 
evidently  to  be  interpreted  by  assuming  that  in  the  neighborhood 
of  the  arsenic  receptor  group  other  groups  arise  or  disappear  and 
thus  cause  the  affinity  to  be  reduced.  The  following  chemical 
example  will  serve  to  illustrate  the  point.  Benzylcyanid  reacts  with 
nitrosodimethylanilin.  In  order  that  the  reaction  take  place,  how- 
ever, heat  and  a  strong  condensing  agent,  free  alkali,  are  required. 
However,  on  introducing  a  nitro  group  into  the  benzole  nucleus,  the 
reactivity  of  the  methylen  group  is  markedly  increased,  so  that  the 
two  substances,  nitro-benzylcyanid  and  nitrosodimethylanilin,  react 
even  in  the  cold.  In  this  case,  therefore,  the  introduction  of  the 
nitro  group  has  exercised  a  quickening  influence  on  the  reaction. 
If,  however,  the  nitro  combination  is  reduced  to  p-amidobenzylcyanid, 
we  find  that  the  latter  is  less  active  than  the  original  material.  The 
amido  group  has  suffered  a  reduction  of  affinity.  The  acetyl  produc 
of  the  amido  combination,  on  the  other  hand,  reacts  to  about  tht 
same  degree  as  the  original  material. 

This  simple  illustration  shows  that  three  different  groups  attaching 
to  the  benzole  nucleus  in  the  para  position  either  increase  the  affinity 
of  the  methylen  group,  or  decrease  it,  or  leave  it  unchanged.  The 
reduction  of  affinity  here  observed  would  correspond  to  the  affinity 
which  we  have  described  above. 

According  to  my  view,  then,  we  should  consider  protoplasm  as 


692  COLLECTED  STUDIES  IN  IMMUNITY. 

made  up  of  a  large  number  of  individual  functions,  which,  in  the 
form  of  different  chemo-receptors  are  scattered  amongst  the  nutri- 
receptors.  I  believe  that  these  two  main  groups  cannot  but  be 
closely  related,  and  for  the  following  reason. 

Trypanosomes  of  different  origin,  as  they  are  cultivated  in 
different  laboratories,  usually  from  the  outset  behave  differently 
toward  a  particular  therapeutic  substance.  The  first  strain  of 
trypanosome  with  which  I  worked,  Mai  de  Caderas,  had  no  resistance 
whatever  against  trypaii  red,  and  this  substance  could  be  employed 
to  effect  a  cure.  This  still  holds  true.  Similar  favorable  results 
were  obtained  by  Jakimoff  in  Russia,  while  Uhlenhuth  obtained 
absolutely  no  result  with  this  substance  on  the  strains  which  he  used. 
We  are  therefore  dealing  with  natural  differences  in  the  various 
strains.  Despite  the  fact  that  my  strain  has  now  been  passed  through 
normal  mice  for  many  years,  it  can  still  be  cured  by  trypan  red  just 
as  well  as  ever.  This  shows  that  the  difference  is  not  entirely 
artificial.  On  the  other  hand,  my  Nagana  strain  could  formerly 
not  be  healed  by  trypan  red,  and  cannot  be  healed  by  that  substance 
now.  However,  on  transforming  this  Nagana  strain  into  a  relapse 
strain,  we  were  surprised  to  find  that  this  property,  which  had  per- 
sisted for  many  years,  become  altered  within  14  days.  This  proves 
that  the  chemo-receptors  really  are  related  to  the  constitution  of 
the  protoplasm,  and  undergo  alterations  when  we  alter  the  con- 
stitution of  the  protoplasm. 

Whether  the  reverse  holds  true,  that  is,  whether,  by  influencing 
the  chemo-receptors  we  can  alter  the  cell  substance,  particularly  the 
nutri-receptors,  has  not  yet  been  definitely  decided.  Browning,  to 
be  sure,  has  observed  that  by  means  of  serum  reactions  one  can 
differentiate  the  fuchsin  strain  from  the  atoxyl  strain,  and  both  from 
the  original  strain.  Careful  investigation  subsequently  showed, 
however,  that  the  changes  in  question  were  not  specific  alterations 
related  to  the  fuchsin  or  to  the  arsenic,  but  alterations  which  cor- 
respond to  the  relapse  mutation  described  above.  These  are  due  to 
the  fact  that  during  the  treatment  it  often  happens  that  the  mice 
suffer  relapses,  which  in  turn  lead  to  the  formation  of  relapse  strains. 

This  brings  me  to  the  close  of  my  paper.  I  am  well  aware  that 
what  I  have  offered  you  has  been  quite  fragmentary,  but  this  could 
hardly  be  otherwise,  for  the  adequate  discussion  of  this  theme  would 
mean  the  recapitulation  of  an  almost  endless  amount  of  work.  My 
object  in  presenting  this  subject  has  been  to  show  you  that  we  are 


THE  PARTIAL-FUNCTIONS  OF  CELLS.  693 

gradually  approaching  the  problem  of  securing  an  insight  into  the 
nature  of  the  action  of  drugs.  I  hope,  too,  that  a  systematic  appli- 
cation of  the  views  I  have  here  presented  will  facilitate  a  rational 
development  of  the  science  of  drug  synthesis.  In  this  connection 
I  may  say  that  thus  far  arsenophenylglycin  has  proven  in  animal 
experiments  to  be  a  truly  ideal  therapeutic  agent.  By  the  aid  of 
this  substance  it  is  possible  to  completely  cure  every  kind  of  trypano- 
some  infection  in  any  kind  of  animal,  and  that  by  means  of  but  a 
single  injection.  Truly,  such  a  result  may  be  termed  therapia 
sterilisans  magna. 


INDEX    OF   ILEMOLYTIC   AND  BACTERIOLYTIC 
REACTIONS   DESCRIBED   IN    TEXT 


AMBOCEPTOR 

Chicken  >  vibrio  Metchnikoff 

Goat  >  sheep  serum 

Goat  >  sheep  blood 

Goat  >  sheep  blood 

Goat  >  sheep  blood 

Goat  >  ox  blood 

Goat  >  ox  blood 

Goat  >  goat  blood 

Goat  >  dog  blood 

Goat  >  rabbit  blood 

Goat  >  vibrio  Xordhafen 

Goat  >  vibrio  Xordhafen 

Goose  >  vibrio  Metchnikoff 

Goose  >  vibrio  Metchnikoff 

Goose  >  ox  blood 


Guinea-pig  >  vibrio  cholera 
Guinea-pig  >  rabbit  blood 
Guinea-pig  >  rabbit  blood 

Guinea-pig>cow  milk 

Guinea-pig  >  rabbit  blood 

Rabbit  >  ox  blood 

Rabbit  >  goat  blood 

Rabbit  >  ox  blood 


Rabbit  >  cow  milk 

Rabbit  >  cow  milk 

Rabbit  >  ox  blood 

Rabbit  >  goat  blood 

Rabbit  >  ox  blood 

Rabbit  >  goat  blood 

Rabbit  >  goat  blood 

Rabbit  >  ox  blood 

Rabbit  >  ox  blood 

Rabbit  >  ox  blood 


CELLS 

vibrio  Metchnikoff 

sheep  blood 

sheep  blood 

sheep  blood 

sheep  blood 

ox  blood 

ox  blood 

goat  blood 

dog  blood 

rabbit  blood 

vibrio  Nordhafen 

vibrio  Nordhafen 

vibrio  Metchnikoff 

vibrio  Metchnikoff 

ox  blood 


vibrio  cholera 
rabbit  blood 
rabbit  blood 
ciliated  eipthelium 

rabbit  blood 

ox  blood  or  goat  blood 

ox  blood  or  goat  blood 

ox  blood 


ciliated  epithelium 

ox  blood 

ox  blood 

goat  blood 

ox  blood  or  goat  blood 

ox  blood  or  goat  blood 

Goat  blood 

ox  blood 

ox  blood 

sheep  blood 


COMPLEMENT 

chicken 
goat  or  sheep 

goat 

goat  or  horse 
horse 


PAGE 
133 
4 
13 
69 
65 
107 
197 
26 
198 
197 
123 
124 
135 
136 


goat 
goat 
goat 
goat 
goat 

guinea-pig 
rabbit  or  pigeon 

goose 

guinea-»pig,  rabbit,  rat, 
goose,  chicken,  goat, 
pigeon,  horse,  115 

guinea-pig  1 

guinea-pig  2 

rabbit  or  guinea-pig        68 
rabbit  53 

rabbit  or  guinea-pig       68 
rabbit  94 

guinea-pig  96 

guinea-pig,  rabbit,  rat, 
goose,  chicken,  goat, 
pigeon,  horse,         115 
rabbit  53 

rabbit  53 

rabbit  53 

guinea-pig  76 

guinea-pig  94 

guinea-pig  96 

goat  104 

rabbit  159 

guinea-pig  597 

guinea-pig  602 

695 


696      INDEX   OF    H^MOLYTIC    AND   BACTERIOLYTIC    REACTIONS 


Rabbit  >  pig  blood 
Rabbit  >  vibrio  Metchinikoff 

Sheep  >  dog  blood 
Inactive  normal  goat  serum 
Inactive  normal  goat  serum 
Inactive  normal  dog  serum 
Inactive  normal  dog  serum 
Inactive  normal  dog  serum 


pig  blood 
vibrio  Metchnikoff 

dog  blood 

rabbit  blood 

guinea-pig  blood 

guinea-pig  blood 

guinea-pig  blood 

rabbit  blood 


guinea-pig 

rabbit 

sheep  or  goat 
horse 
horse 
guinea-pig 
horse 
horse 


602 

122 

76 

59,  65 
65 
60 
60 
64 


NOTE. — For  reactions  showing  the  joint  action  of  several  amboceptors  see 
pages  601  and  616;  for  reactions  with  active  normal  sera,  see  subject  index 
under  the  respective  animal;  for  reactions  involving  antilytic  sera,  see  subject 
index  under  Anticomplements,  Antihcemolysins,  AntiawJboceptors. 


INDEX  OF  AUTHORITIES  QUOTED 


Abel,  120 

Arloing,  143,  144 

Aronson,  406,  407 

Arrhenius,  S.,  72,  513 

Arrhenius  and  Madsen,  481,  484,  486, 

489,  502,  515,  552,  578 
Asakawa,  318 
Atkinson,  J.  P.,  145 

Babes,  143,  146 

Baeyer,  437 

Bail,  589,  298,  318,  541 

Bashford,  71,  490,  532 

Baumann,  407 

Baumgarten,  236 

Bayer,  430 

Bechhold  and  Ehrlich,  442 

v.  Behring,  357,  358,  364,  519 

Behring,  656,  677 

Belfanti  and  Carbone,  23,  26,  379,  392 

Bertrand,  G.,  180 

Besredka,  249,  283,  285,  532,  601 

Bier,  332 

Bing,  306 

Blumenthal,  356,  359 

Bohm,  406 

Bolton,  541,  591 

Bordet,  J.,  1,  2,  3,  4,  13,  20,  21,  26,  36, 
52,  56,  58,  63,  64,  67,  68,  69,  71,  72, 
74,  77,  78,  88,  111,  131,  142,  171,  181, 
195,  196,  201,  204,  300,  378,  381,  469, 
512,  541,  562,  565,  588,  598,  616,  650 

Bordet  and  Gay,  617,  629 

Bordet  and  Gengou,  349 

Bordet  and  Malkow  392 

Borrel,  47,  71 

Braun,  405 

Brieger,  406 

Briot,  479 

Browning,  580,  618,  631,  649,  650,  687 


Browning  and  Sachs,  649 

Bruno,  421 

Brunton,  406 

Buchheim,  425 

Buchner,  13,  15,  21,  56,  58,  59,  62,  74, 
88,  94,  118,  182,  183,  191,  195,  208* 
210,  217,  364,  386,  394,  587,  588 

Bulloch,  94,  333,  340 

Bunsen,  689 

van  Calcar,  555,  577 

Calmels,  175,  180 

Calmette,  293,  301,  302,  311,  403,  444, 

466,  478 

Camus  and  Gley,  20,  539 
Capparelli,  180 
Carbone,  23 
Celli,  324 
Cnyrim,  223 
Cobbett,  541,  591 
Conradi,  317,  282 
Courmont,  91 
Courmont  and  Doyon,  536 
Creite,  3 

Danysz,  356,  374,  482,  516,  556,  671 

Decroly  and  Rousse,  294,  535 

Delbriick,  231 

Deutsch,  375 

Dewar,  407 

Dieudonne,  454 

Donath  and  Landsteiner,  581 

Donitz,  64,  91,  117,  118,  216,  359,  420 

Dreyer  and  Madsen,  507,  509,  522,  531 

Drigalski  and  Conradi,  317 

Duden,  431 

v.  Dungern,  21,  23,  24,  36,  47,  56,  62, 
64,  68,  74,  93, 100, 118,  146,  156,  158, 
160,  161,  201,  213,  242,  243,  250,  378, 
556,  671 

697 


€98 


INDEX    OF    AUTHORITIES    QUOTED 


Durham,  89,  378,  385,  599 
Duval,  313 

Ehrlich,  P.,  5,  8,  15,  21,  43,  47,  51,  52, 
64,  74,  77,  82,  89,  100,  129,  131,  137, 
138, 143, 146, 158,  162, 164, 167, 171, 
176, 180, 182, 196,  215,  216,  233,  242, 
254,  284,  301,  316,  360,  365,  390,  391, 
398,  404,  442,  452,  577,  588,  591,  676 

Ehrlich  and  Marshall,  226,  286,  309, 
638 

Ehrlich  and  Michaelis,  408 

Ehrlich  and  Morgenroth,  1,  llr  23,  36, 
43,  47,  56,  71,  88,  127,  130,  131,  132, 
167, 179, 181, 182, 188, 196,  205,  209, 
219,  225,  226,  243,  284,  288,  291,  298, 

.   334,  588,  595,  598,  602,  605,  616,  626 

Ehrlich  and  Sachs,  195,  209,  213,  226, 
228,  309,  444,  547,  561,  617,  620,  625, 
628,  634,  650,  652,  660 

Einhorn,  407,  439 

Eisenberg,  318 

Eisenberg  and  Volk,  298,  318,  341 

Elf  strand,  335,  391 

Falk,  439 

Filehne,  406,  407 

Fischer,  E.,  2,  532,  678 

Fleischmann  and  Michaelis,  657 

Flexner,  313,  541 

Flexner  and  Noguchi,   194,  291,  293, 

300,  339,  443,  456,  459,  467,  581 
Fliigge,  587 
Frankel,  534 
Frankel,  E.  and  Otto,  4 
Francke,  687 
Fraser  and  Braun,  405 
Friedberger,  156,  341,  564,  582,  601, 

608,  610,  651 
Friedemann,  265,  346,  353 
Fuhrmann,  580 

Gabriel,  429 

Galeotti,  438 

Gay,  580,  584,  585,  610,  617 

Gengou,  349,  584,  585,  611,  649 

Geppert,  425 

Gerlach,  438 

Gibbs,  406 

Gley,  20 

Goldscheider,  416 

Graebe  and  Liebermann,  411 


Gruber,  M.,  9,  134,  138,  140,  142,  182, 
188,  191,  215,  219,  220,  225,  233,  234, 
235,  250,  265,  358,  378,  391,  514,  534 

Gruber  and  Durham,  599 

Gulbransen,  683 

Harnack,  425 

Hedon,  490 

Henriques  and  Bing,  306 

Hinsberg,  407 

Hirschlaff,  532 

van't  Hoff,  72 

Hofmeister,  231,  426 

Jacoby,  487 

Jaffe,  406 

Jakimoff,  692 

Jornara  and  Casali,  175,  180 

Kehrman  and  Baeyer,  437 

Kendrick,  407 

Kitashima,  357,  358 

Klein,  268,  618,  624 

Kleine,  683 

Knecht,  434 

Knorr,  51,  214,  504,  679 

Robert,  173,  175,  391 

Koch,  688 

Kolle,  4 

Koppe,  560 

Korschun,  267,  281,  340,  517,  597 

Korschun  and  Morgenroth,  267,  340, 

597 

Kossel,  20,  401,  539 
Krafft,  418 

Kraus,  R.,  318,  378,  588,  681 
Krauss,  591 
Kretz,  143,  145 
Kruse,  312 

Kyes,  291,  457,  460,  467,  484,  581 
Kyes  and  Sachs,  443 

Ladenburg,  440 

Lamb,  300,  478 

Landois,  3 

Landsteiner,  23,  24,  581,  591,  599 

Landsteiner  and  Sturli,  284 

Leclainche  and  Morel,  121 

Levaditi,  431,  265 

Liebermann,  411 

Liebreich,  439 

v.  Lingelsheim,  391 


INDEX    OF    AUTHORITIES    QUOTED 


699 


Lipstein,  132, 220, 226, 265, 316, 355, 575 

Loffler  and  Abel,  120 

London,  111,  182,  194,  249 

Low,  427,  428 

Lubowski,  R..  146,  156,  158,  161 

Madsen  and  Walbaum,  558 

Madsen,  143,  145,  217,  330,  366,  391, 
481,  484,  488,  507,  509,  522,  552,  656, 
658,  673 

Magendie,  332 

Malkow,  62,  88,  392,  590 

Mannaberg,  418 

Marie,  71,  356 

Markl,  214,  329,  337 

Markwald,  430 

Marshall,  222,  226,  228,  246,  286,  309, 
335,  566 

Marshall  and  Morgenroth,  228,  283, 
286,  566 

Martin,  489 

Martin  and  Cherry,  466,  558 

Matthes,  163.  164,  165,  166 

Marx,  5,  348,  356,  375 

Meltzer,  386 

Mering,  406 

Mertens,  328,  348 

Mesnil,  683 

Meyer,  Hans,  427 

Meyer,  R.,  411 

Metalnikoff,  83,  87,  193 

Metchnikoff,  E.,  1,  24,  45,  46, 48,  51,  71, 
72,  91,  111,  118,  136,  137,  208,  220, 
267,  271,  272,  275,  356,  375 

Michaelis,  408,  657 

Michaelis  ard  Fleischmann,  658 

Miescher,  402 

Milchner,  356 

Moll,  281 

Morel,  121 

Moreschi,  584,  585,  611,  649,  651,  657, 
660 

Morgenroth,  1, 11,  23,  36,  43,  47,  56,  64, 
71,  88,  91,  92, 127,  130, 131,  132, 163, 
167,  179,  181,  182,  188,  189,  196,  209, 
219,  225,  226,  228,  241,  243,  250,  267, 
283,  284,  288,  291,  298,  326,  333,  378, 
391,  566,  588,  591,  595,  669 

Morgenroth  and  Sachs,  233,  250,  595, 
609,  618 

Moxter,  24,  39,  49,  58,  193,  242 

Muir,  580,  650 

Muir  and  Browning,  650,  652 


Muller,  P.  Th.,  81,  111,  118,  182,  192, 

239,  249,  265,  288,  333,  339,  346 
Myers,  209,  295,  467,  473,  487 

Xeisser,  E.,  and  Doring,  182,  205 
Neisser,  E.,  and  Freidmann,  265,  346, 
Neisser,  M.,  88,  117,  146,  317,  349,  587 
Neisser  and  Lubowski,  146,  156,  158, 

353 

Neisser  and  Sachs,  611,  659 
Neisser  and  Wechsberg,  82,  120,  132, 

133,  134,  136,  137,  142,  193,  220,  226, 

256,  295,  313,  348,  381,  461,  469,  566, 

606 

Nencki,  232,  406 
Nernst,  559 
Nicolle,  74 

Nicolle  and  TrSnell,  147 
Nietzki,  412 
Nissen,  589 
Nissl,  416 
Noguchi,  194,  291,  293,  300,  339,  443, 

455,  456,  459,  467,  581 
Nolf,  74,  118,  171 
Nuttall,  589 

Obermayer  and  Pick,  579 

Ostertag,  289 

Ostwald,  410 

Otto,  4 

Otto  and  Sachs,  656 

Overton,  427,  436,  532 

Paltauf,  358,  542 

Park  and  Atkinson,  145 

Pasteur,  500 

Pavlovsky,  143 

Penzoldt,  407 

Pfeiffer,  R.,  1,  2,  4,  39,  120,  193,  250, 

378,  541 
Pfeiffer  and  Friedberger,  156,  341,  564, 

582,  601,  608,  610,  651 

161,  321 

Pfeiffer  and  Kolle,  4 
Pfeiffer  and  Marx,  5,  348,  375 
Pfeiffer  and  Moreschi,  651 
Pfluger,  398 
Phisalix,  455,  466 
Phisalix  and  Bertrand,  180 
Pick,  579 

v.  Pirquet,  514,  527 
v.  Pirquet  and  Eisenberg,  318 
Plimmer,  690 


700 


INDEX    OF    AUTHORITIES    QUOTED 


Pohl,  71  ,  426,  490,  532 
Poulson,  439 
Proscher,  175 
Pugliese,  A.,  175,  180 

Ransom,  356,  358,  374,  376,  543 

Rehns,  94,  143,  145,  147,  161,  332,  421 

Rohl,  683,  687 

Romer,  290,  374,  578 

Rousse,  294,  535 

Roux,  91,  359,  376,  420,  541 

Roux  and  Borrel,  47,  71 

Roux  and  Vaillard,  366,  542 

Sachs,  H.,  138,  146,  156,  158,  163,  167, 
181,  195,  209,  210,  220,  222,  233,  234. 
250,  309,  340,  443,  547,  561,  601,  610, 
616,  617,  620,  625,  628,  634,  650,  652, 
656,  660,  673 

Sachs  and  Bauer,  616 

Salomonsen  and  Madsen,  366 

Schattenfroh,  244,  289 

Schmiedeberg,  425,  426 

Schonlein,  21 

Schreiber,  290 

Schutze,  334,  585 

Schutze  and  Scheller,  205 

Sclavo,  503 

Shibayama,  268,  271 

Shibayama  and  Toyoda,  652 

Shield,  146 

Shiga,  312 

Sobernheim,  117 

Spiro,  426,  437 

Spronck,  18 

Stahlschmidt,  405 

Stas-Otto,  426 

Stephens  and  Myers,  295 


Straub,  680 
Sturli,  284 

Takaki,  360 

Tarassevitch,  268,  271,  272,  275 

Tizzoni,  467,  519 

Toyoda,  652 

Trenell,  147 

Tschistovitsch,  243,  681 

Uhlenhuth,  334,  585,  681,  692 

Vaillard,  366,  542 
Vedder  and  Duval,  313 
van  de  Velde,  391 
Verworn,  397,  398 
Virchow,  364,  387 
Vulpian,  180 

Walbaum,  558 

Wassermann,  A.,  5,  77,  118,  222,  356, 

359,  375,  585,  594,  681 
Wassermann  and  Ostertag,  289 
Wassermann  and  Schutze,  334 
Wassermann  and  Takaki,  360 
Wechsberg,  82,  83,  120,  132,  133,  134, 

136, 137,  138,  142,  193,  215,  220,  222, 

226,  256,  295,  313,  348,  353,  381,  391, 

461,  469,  566,  591 
Weigert,  C,  9,  47,  90,  92,  100,  537 
Welch,  546 

Wendelstadt,  205,  222  236 
Widal,  385,  391,  681 
Widal  and  Sicard,  4 
Wilde,  201 
Witt,  412,  434 

Zupnik,  356 


INDEX   OF  SUBJECTS 


NOTE. — The  numbers  printed  in  bold  face  type  refer  to  pages  on   which   the 
topic  is  specifically  discussed. 

PAGE 

Absorption,  elective 16,  59,  97 

mechanical,  contrasted  with  chemical  union 78 

of  a  serum  by  its  antigen 6 

of  complement  (see  also  Deflection  of) ' 585 

of  complement  by  cellular  material 201 

of  complement,  by  sensitized  cells 196 

Absorption  test,  to  demonstrate  multiplicity  of  antibodies 590 

Abrin,  local  immunity  against 375 

Acid,  influence  of,  on  complement 199 

Addiment  (complement) 4 

Additive  properties,  of  chemical  groups 410 

Adsorption,  as  factor  in  lysin  action 74 

in  relation  to  complements 200 

lack  of  specificity 78 

Affinity,  between  cell  and  amboceptor 218 

between  diphtheria  toxin  and  antitoxin 484 

changes  in,  in  complementoid  formation 82 

changes  in,  in  immune  body 127 

changes,  in  of  haptophore  groups 209 

importance  of  changes  in 580 

of  cells  for  immune  body 75 

of  complement,  immune  body  and  erythrocytes 8 

relative,  of  tissue  receptors  and  injected  cells 162 

Age,  influence  exerted  by,  on  antitoxic  sera 675 

Agglutination,  effect  of  heat  on 2 

of  sheep  blood  by  goat  serum 3 

relation  to  haemolysis 4 

in  deflection  of  complement 126,  134 

Agglutinins,  as  distinct  antibodies 4 

Aleuronat,  character  of  exudates  produced  by 44 

Alexin 56 

action  of 181 

ferment  character  of 58 

Alkali,  influence  of,  on  complement 198 

Amboceptor,  complementophile  groups  of 227 

enormous  quantity  absorbed  by  cholera  vibrios 157 

first  use  of  the  term Ill 

701 


702  INDEX    OF    SUBJECTS 

PAGE 

Amboceptor,  occasional  slight  affinity  for  cell  receptors 580 

of  dog  serum,  thermolability  of 210 

plurality  of 574 

quantitative  relation  to  complemen.t 250 

saturation  of  blood-cells  with 159 

Amboceptors,  against  dissolved  albumins .f 585 

complementibility  of 233 

hsemolytic,  in  response  to  serum  injections 211 

hsemolytic,  the  binding  of 595 

mechanism  of  their  action 209 

normal  and  immune 233 

Amboceptor  union,  dissociation  of 596 

Amboceptor  unit,  definition  cf 254,  595 

Anaesthetic  action,  chemical  relations  of 407 

Animal,  choice  of,  in  production  of  anticomplement  sera 66 

Animal  individuality,  expressed  in  isolysins 30 

Anthropostable  complements 43 

Antialexin  (see  Anticomplement). 

Antiamboceptors,  mode  of  action 561 

production  of 333 

studies  on 649 

Antiantiamboceptors : 651 

Antiantolysin ;  .  .     33 

Antibacteriolytic  action,  of  normal  serum 601 

Antibodies,   against  bacteriolysins  and  hsemolysins 64 

in  normal  serum,  multiplicity  of 587 

multiplicity  of 384 

normal 587 

varieties  possible  by  immunization 24 

Antibody,  formation  of,  various  phases 90 

Anticomplement 63 

choice  of  animal  in  production  of 66 

isogenic  and  alloiogenic 260 

mode  of  action 65 

quantitative  relation  to  complement 258 

rabbit>  goat 20 

Anticompliments,  against  serum  of  horse,  goat,  dog,  ox,  rabbit,  and  guinea- 
pig 66 

as  cause  of  deflection  of  complement 133,  136,  138 

as  thrust-off  amboceptors 225 

in  Pfeiffer-Friedberger  phenomenon 603 

partial 222 

produced  by  immunization 64 

production  of 333 

protection  afforded  by  various 1 14 

really  free  amboceptors 605 

Anticomplementary  serum,  polyvalence  of 66 

Antiferments,  in  normal  sera 591 

Antihaemolysins 64,  102,  114,  258,  333,  342,  649 

method  of  study 342 

natural 283 

see  also  anticomplements,  and  antiamboceptors 561 

Anti-immune  body,  character  of 101,  105 


INDEX    OF    SUBJECTS  703 

PAGE 

Anti-immune  body,  specificity  of 109 

normal 102 

Anti-isolysin 28 

Antilysin,  against  eel  serum 20 

against  toad  poison 179 

multiplicity  of 20 

Antipyretic  .action,  chemical  relations  of 407 

Antisperma toxin 52,  72 

Antitoxic  serum,  complex  character  of 368 

genetic  method  of  study 368 

Antitoxin,  complex  character  of 368 

disproportion  in  production  of,  to  amount  of  toxin  injected 679 

in  normal  horses 541 

in  normal  sera 591 

occurrence  in  normal  individuals 367 

site  of  origin 375 

supposed  to  be  transformed  toxin 366 

Antitoxins,  source  of 48 

Straub's  conception  of  action  of. 680 

Antitryptic  substances,  in  normal  serum 591 

Apes,  use  of,  for  obtaining  sera 117 

Arachnolysin,  antitoxin  against : 173 

properties  of 169 

Arsenic-fast  trypanosomes 687,  690 

Atoxyl,  a  trypanocidal  agent 688 

Atrepsy,  a  form  of  immunity 684 

Autoanticomplement 83 

Autolysin,  definition  of 27 

Bacillus,  of  dysentery 312 

Bactericidal  experiments,  technique 384 

Bactericidal  serum,  action  of 120 

Bacteriolysins,  side-chain  theory  applied  to 5 

Bacteriolysis,  relation  to  agglutination 4 

its  similarity  to  haemolysis 2 

Metchnikoff's  demonstration  of,  in  vitro 1 

Pfeiffer's  theory  of 1 

regarded  as  a  ferment  action 2,  8 

substances  concerned  in 4 

Biogens 398 

Bleeding,  of  animals,  for  serum 349 

Blocking,  by  complementoid 345 

Blood,  protective  substances  in 364 

Blood-cells,  as  food  storages 402 

Blood-cells,  behavior  toward  cobra  venom 292 

discoplasm,  function  of 171 

function  of,  in  nutrition 397 

hardened,  haemolysis  of 163 

lecithin  content  of  stroma  of 449 

receptor  apparatus  of 390 

stroma  of,  to  bind  immune  body 74 

varying  susceptibility  to  cobra  venom 458 

Bone  marrow,  as  source  of  immune  bodies 5 


704  INDEX    OF    SUBJECTS 

I'AOB 

Bordet-Gengon,  phenomenon  of 196 

Bordet's  sensitization  theory,  contrasted  with  Ehrlich's  amboceptor  theory. .  58 

Bovine  serum  (see  also  Ox  serum),  effect  on  guinea-pig  blood 18 

Brain  tissue,  power  to  neutralize  tetanus  toxin 356 

union  with  tetanus  toxin 5 

Bufidin 175 

Cancer,  treatment  with  lactoserum 55 

Castration,  effect  of,  on  production  of  spermotoxin 48 

Cell  immunity,  without  formation  of  antibodies 539 

Cells,  partial  functions  of 676 

Chemical  constitution,  relation  to  pharmacological  action 404 

Chemical  distribution,  relation  to  pharmacological  action 415 

Chemical  nature,  of  haemolytic  action 6 

Chemical  nature,  of  immunity  reaction 78 

Chemical  poisons,  action  of 532 

Chemical  union,  prerequisite  for  formation  of  antibody 5 

Chemoreceptors 686 

Chicken  serum,  action  on  rabbit  blood 192 

Cholera,  bacteriolysis  of  vibrios  of 1 

Cholera  immune  bodies,  source  of 5 

Cholesterin,  action  in  cobra- venom  haemolysis 454 

Ciliated  epithelium,  from  ox  trachea,  method  of  collection 49 

Cobra  lecithid,  absence  of  neurotoxic  action  of 472 

properties  of 470 

Cobra  venom,  studies  on 291 

substances  which  activate 443 

Coctostable,  definition  of  the  term 340 

hsemolytic  organ  extracts 281 

Colligative  properties,  of  chemical  groups 410 

Colloid  chemistry,  applied  to  immunity  reaction 578 

Colloide  de  bceuf,  of  Bordet-Gay 619 

Combining  capacitv,  of  cells  for  amboceptors 396 

Common  receptors 95 

in  tracheal  epithelium,  blood-cells,  in  other  tissues,  38,  49,  51 

Complement,  absence  of  direct  affinity  for  erythrocytes 6 

absorption  by  yeast 213 

action  of 181 

deflection  of 120,  132 

deflection  of,  power  of  normal  serum  to  produce 610 

deflection  of,  role  of  precipitates  in 611,  651,  656 

dominant  and  non-dominant 227,  618 

effect  of  phosphorus  poisoning  on 63 

Ehrlich's  original  Unitarian  conception  of 9 

finding  additional  sources  of 117 

first  use  of  the  term  by  Ehrlich 16 

from  different  animals 115 

fixation,  Bordet-Gengou 196 

homostable 117 

influence  of  purulent  process  on  production  of 87 

influence  of  various  agents  on 198 

in  spleen 44 

in  phagocytes 44 


INDEX    OF    SUBJECTS  705 

PAGE 

Complement,  its  union  with  amboceptor  alone 580 

loose  union  with  immune  body 8 

method  of  measuring  amount 38 

not  increased  by  immunization 39 

Complementibility,  fluctuations  of,  of  an  immune  serum  by  different  com- 
plements   69 

of  various  interbodies 191 

Complementoids,  action  of 79,  209 

blocking  complements 345 

existence  of 580 

Complementophile  group,  structure  of 582 

Complements,  anthropostable 43 

behavior  toward  Pukall  filters 59 

constitution  of 65 

differentiation  of,  by  partial  anticomplements 222 

disappearance  of,  under  natural  circumstances 86 

methods  of  preserving 329 

multiplicity  of 15,  110,  195,  222,  382 

of  horse  serum 239 

partial 114 

quantitatively  independent  of  immune  body 38 

quantitative  relation  to  amboceptor  and  anticomplement .  .  .  258 

relation  to  phagocytes 43 

similarity  of  majority  of,  in  certain  species 66 

thermostable,  in  goat  serum 13 

thermostable,  in  sheep  and  calf  serum 15 

various  cells  which  absorb 41 

Constitutive  properties,  of  chemical  groups 410 

Copula  ( =  immune  body) Ill 

Cross  absorption,  in  study  of  common  immune  bodies 97 

Crossed  immunization,  and  reciprocal  elective  absorption 97 

Cytase  (  =  complement) Ill,  267 

Danysz,  effect  of 671 

Deflection  of  complement 120,  132,  584 

by  normal  serum 610 

due  to  precipitates 611,  651,  656 

in  cobra-venom  haemolysis 469 

role  of  agglutination 126,  134 

Desmon  (  =  immune  body) Ill 

Deuterotoxoid 497 

Diazobenzaldehyd,  function  of  its  side-chains 73 

Digestion,  haemolysis  analogous  to 8 

Diphtheria  antitoxin,  heating  of 18 

Diphtheria  bacillus,  poisons  produced  by 512,  548 

Dipththeria  toxin,  constituents  of 481 

Discoplasma,  of  blood-cells,  function  of 171 

Dissociation,  in  toxin-antitoxin  combination 666 

of  agglutinin  combination 599 

of  amboceptor  union 599 

Distribution,  chemical,  in  organism 410 

Distributive  property,  importance  of 415 

Dog  blood,  behavior  toward  arachnolysin 170 


706  INDEX    OF   SUBJECTS 

PAOB 

Dog  serum,  action  on  guinea-pig  blood 210 

action  on  cat  blood 21 

action  on  guinea-pig  blood 18 

effect  of  heat  on  its  haemolytic  power 18 

fluctuation  in  its  hsemolysins 21 

thermolability  of  its  complement 187 

Dominant  and  non-dominant  complements 618 

Dosage,  of  bactericidal  sera,  paradoxical  results 120 

Dyeing,  compared  to  binding  of  lysins 75 

Dysentery,  bacillus  of   studies  on 312 

Eel  serum  (see  Ichthyotoxin) 19 

Ehrlich's  first  classical  experiments  on  haemolysis 5 

Ehrlich's  phenomenon  (toxin-antitoxin) 485 

Ehrlich's  Side-chain  Theory 5 

Elective  absorption,  in  study  of  common  immune  bodies 59,  97 

Endocomplements 295,  443 

action  due  to  lecithins 451 

Epithelium,  ciliated,  how  collected 49 

immune  serum  against 24,  48 

Epitoxoid 503 

Erythrocytes  (see  also  under  Blood,  and  under  Individual  animals). 

mammalian,  their  side-chains 43 

-receptor  apparatus  of 390 

stromata  of 171 

Ethyl  green,  as  trypanocidal  agent :  . .  .  687 

Exhaustion,  of  a  specific  serum  by  its  antigen 6 

Fatty  acids,  hsemolytic  action  of 464 

Ferment  action,  its  similarity  to  bacteriolysis 2,  8 

Fixation  reaction,  Bordet-Gengou 196 

Fluctuation  in  ha3molytic  power  of  sera 21 

Fluctuation  in  serum  constituents 21 

Fractional  addition  of  blood-cells,  in  haemolysis 599 

Fractional  neutralization,  in  study  of  diphtheria  toxin 481,  552 

Fractional  saturation,  Bordet,  in  study  of  lysins 75 

Frogs,  Courmont's  experiments  with  tetanus  of 91 

Gelatine  filtration,  in  study  of  toxjn-antitoxin 558 

Goose  serum,  immune,  against  ox  blood 115 

immune,  against  vibrio  Metchnikoff 135 

Goat,  complement  of,  ability  to  substitute  sheep  complement  for 66 

immunization  against  goat  blood 26 

Goat  serum,  fluctuation  in  its  haBmolysins 21 

normal,  effect  on  sheep  blood 3,  12 

normal,  effect  on  rabbit  and  guinea-pig  blood 12,  59,  65 

normal,  effect  on  various  bloods 590 

Group  haemolysins,  of  guinea-pig  >  rabbit  serum 2 

Guldberg-Waage  law,  in  toxin-antitoxin  reaction 482,  559 

Haemoglobinuria  ex  frigore 15 

Haemolysin,  compared  to  toxin  molecule 57 

normal,  nature  of 16 

of  cobra  venom .  292 


INDEX    OF    SUBJECTS  707 

PAGE 

Hsemolysin,  thermostable 13 

Haemolysins,  complex  nature  of 3 

complex,  study  of 336 

method  of  studying 326 

multiplicity  of,  in  normal  serum 19 

toxicity  of •  •  •  23 

Haemolysis,  by  arachnolysin 167 

by  joint  action  of  several  amboceptors 616 

by  saponin  poison 478 

Bordet's  studies  on,  applied  to  bacteriolysis : . .  2 

chemical  character  of  the  reaction 73 

effect  of  heat  on 2 

Ehrlich  and  Morgenroth's  first  study  of 3 

of  hardened  erythrocytes 163 

relation  of  osmotic  tension  to 236 

substances  concerned  in 4 

Haemo ly tic  amboceptors,  binding  of 595 

in  response  to  serum  injections 241 

in  response  to  injections  of  urine 244 

Haemolytic  experiments,  method  of  making 330,  334 

Haemolytic  power,  fluctuation  of,  of  normal  serum 238 

Haemolytic  properties,  of  organ  extracts 267 

Haptins,  definition  of 62 

multiplicity  of -..-.• 20,  384 

Heat,  effect  of,  on  diphtheria  antitoxin 18 

effect  on  haemolytic  power 2 

effect  of,  on  immune  serum 4 

effect  on  immune  body-complement  combination 8 

effect  on  normal  hsemolysins 12 

effect  of,  on  serum 631 

hi  inactivation,  care  in  employment  of 187,  192 

Hemitoxin 494 

Hen  serum  (see  Chicken) •. 192 

Heterolysin,  definition  of 27 

Hilfskorper  (Buchner) 182,  387 

Horse  complement,  for  inactive  goat  serum 59 

Horse  serum,  complements  of 239 

large  variety  of  complements  in 64 

normal,  effect  on  typhoid  bacilli 589 

normal,  its  hsemolytic  power .t 237 

Horror  autotoxicus * 82 

Hypersusceptibility 521,  666 

Ichthyotoxin,  inability  to  reactivate. 19 

Idiocomplements 86 

Immune  body  (see  also  Amboceptor). 

constitution  of 6 

Ehrlich's  first  studies  on 4 

loose  union  with  complement 8 

manner  in  which  it  combines  with  cells 73 

multiplicity  of 9 

multiplicity  of  complementophile  groups 112 

relation  of  phagocytes  to  production  of 46 


708  INDEX    OF    SUBJECTS 

PAGE 

Immune  body,  quantitatively  independent  of  complement 38 

site  of  production  of 51 

source  of 5 

Immune  serum,  against  spermatozoa,  epithelium,  leucocytes,  and  kidney 

cells 24 

Bordet's  first  studies  on 1 

manner  in  which  it  differs  from  normal 39 

Immunity,  a  phase  of  physiology  of  nutrition 377 

due  to  absence  of  receptors 28 

local,  against  abrin - 375 

of  cells,  without  formation  of  antibody 539 

regarded  as  increase  of  normal  functions 587 

Immunization,  against  blood-cells 12 

against  body's  own  cells 52 

dependent  on  haptophore  group 51 

hsemolytic,  technique  of 331 

with  agglutinated  bacteria 146 

with  modified  proteins 579 

with  overneutralized  mixtures 143,  14ft,  158 

with  sensitized  blood-cells. 41 

Inactivation,  of  immune  sera  by  heat 4 

Incubation  period,  explanation  of 535 

Individuality,  animal,  expressed  in  isolysins 30 

Interbody,  of  normal  sera 16 

conditions  governing  separation  of,  by  absorption 190 

Intravenous  injections,  in  immunization 160 

Isolysin,  Ehrlich's  experiments  on  production  of 26 

"Kalte  Methode,"  elective  absorption  at  low  temperatures 6,  12,  185 

Kidney  cells,  immune  serum  against 24 

L0  and  Lf,  definition  of 143,  368,  485,  549 

Lactoserum 38,  52 

Lamprey  serum,  varying  toxicity  of 21 

Lateral  chains  (see  also  Side-chain) 5 

Lecithin,  and  allied  substances,  action  of 462 

in  blood-cell  stromata 449 

in  cobra-venom,  haemolysis 443 

relation  to  cobra- venom  haemolysis 305 

Lecithids,  of  cobra-venom 470,  581 

of  snake  venom 466 

of  various  snake  venoms 477 

Leistungskern 399 

Leucocytes,  immune  serum  against 24 

Local  immunity,  against  abrin 375 

Lymph  nodes,  as  source  of  immune  bodies 5 

Lysins,  discovery  of 1 

Ehrlich's  studies  on  the  action  of 1 

similarity  of,  to  toxins 57 

Macrocytase,  hasmolytic  ferment 208,  267 

Macrophage,  relation  to  haemolysis 44,  267 

Malachite  green,  as  trypanocidal  agents 687 


INDEX    OF    SUBJECTS  709 

PAGE 

Mass  action,  in  toxin-antitoxin  combination 482,  556 

Mechanical  absorption,  contrasted  with  chemical  union 78 

Mercury,  cells  hardened  with,  their  haemolysis 163 

Milk,  biological  relation  to  epithelial  cells 55 

immune  serum  against 53 

Microcytase  (Metchnikoff ) 208 

Microphages,  relation  to  haemolysis 44 

M.  L.  D 485 

Monotropisin 417 

Multiplicity,  of  antibodies  in  normal  serum 62 

of  blood-cell  receptors 284 

of  complements 15 

of  complement,  analogy  with  ferments 231 

of  haemolysins  in  normal  sera 58 

of  haptins  in  blood 20 

Neisser- Wechsberg,  phenomenon 120 

Neutral  mixtures,  immunization  with 158,  143,  146 

Normal  haemolysins  (see  also  under  Individual  animals) 12,  16 

mechanism  of 192 

Normal  serum,  antibacteriolytic  action  of 601 

deflection  of  complement  by 610 

its  amboceptors 233 

its  spennotoxic  power 193 

multiplicity  of  antibodies  in 587 

Nutrireceptors,  definition  of 682 

Organ  extracts,  haemolytic  properties  of 267 

Ox  serum,  normal,  action  on  typhoid  bacilli 589 

normal,  in  haemolysis  of  guinea-rig  blood 18 

to  complement  typhoid  immune  bodies 118 

Pancreas  extract,  action  on  blood-cells  hardened  with  mercury 163 

Papain,  influence  of,  on  complement 198 

Partial  amboceptors,  method  of  differentiation 574 

Partial  functions  of  cells 676 

Partial  immune  bodies : 97,  105 

Partial  neutralization,  in  study  of  diphtheric  toxin 481,  552 

Partial  saturation  (Bordet),  in  study  of  lysins 75 

Pepton,  injections  of,  to  increase  complement 118 

Pfeiffer,  theory  of  bacteriolysis 2 

Pfeiffer's  phenomenon 1 

Pfeiffer-Friedberger  phenomenon 601 

Phagocytes,  complement  content  of 44 

relation  to  immunity 45 

Pharmacological  action,  relation  to  chemical  constitution 404 

Phases,  in  antibody  formation 90 

Philocytase  ( =  immune  body) Ill 

Phosphorus  poisoning,  effect  on  complement  production 63 

Phrynin „ 175 

Phrynolysin,  antiserum  against 180 

properties  of 179 

mode  of  preparation « 176 


710  INDEX    OF    SUBJECTS 

PAGE 

Pigeon  serum,  as  complement 115,  135 

Plurality,  of  complements  (see  also  Multiplicity) 195 

Polyceptors 112 

Polyvalent  sera 92,  110,  119 

Precipitates,  and  antiamboceptors 651,  656,  663 

as  cause  of  deflection  of  complement. 611,  651,  656 

Preparator 233 

Preservation,  of  complement  sera 329 

Proagglutinoids 319 

Protective  substances,  in  blood 364 

Prototoxoid 497 

Pukall  filters,  in  differentiating  complements 59 

Quadriceptor 112 

Quantitative  estimation,  of  amboceptors,  complement  and  receptors. 340 

Quantitative  relations,  between  amboceptor,  complement,  and  anticomple- 

ment 250 

between  cobra-venom  and  lecithin 456 

between  immune  body  and  complement 38 

Rabbit  blood,  action  of  goat  serum  on 12,  59,  65,  590 

Rabbit  serum,  action  on  goat  blood 245 

fluctuation  in  its  haemolysins 21 

normal,  action  on  various  bacteria 589 

normal,  action  on  ox  blood  haemolysis 606 

normal,  in  haBmolysis  of  sheep  blood  and  goat  blood 18 

Reactivation  of  inactive  immune  sera 4 

Receptors,  absence  of,  as  cause  of  immunity 28 

common. 51,  95,  242 

definition  of 24 

of  blood-cells 390 

nature  of 241 

sessile 92 

specificity  of 100 

various  orders  of 392 

Rennin,  immunization  against 8,  92 

simultaneous  occurrence  of  rennin  and  antirennin  in  body 32 

Reversible  reaction,  in  amboceptor  combination 596 

in  toxin-antitoxin  combination 555 

Saponin,  action  of 455,  478 

Salts,  action  of,  in  haemolysis * 213 

Sensitization  theory 37,  67,  68,  131,  381,  469,  562,  579 

contrasted  with  amboceptor  theory 58 

regarded  from  chemical  or  biological  standpoints 63 

Sensitizer  (or  amboceptor?) 217 

Serum  (see  also  under  Individual  animals). 

bactericidal,  mode  of  action  of 120 

collecting  and  preserving  for  hsemolytic  work 326 

collecting  of,  for  bactericidal  tests 349 

Serum-fast  strains  of  trypanosomes 684 

Sessile  receptors 92 

Sequence  of,  importance  of,  in  deflection  experiments 658 


INDEX    OF    SUBJECTS  711 

PAGE 

Sheep  blood,  agglutination  of,  by  goat  serum 3 

Sheep  complement,  substitution  of,  for  goat  complement 66 

Sheep  serum,  normal,  in  haemolysis  of  guinea-pig  blood 18 

Side-chains,  constitution  of  various  kinds  of 9 

physiological  object  of 20 

their  primary  function 9 

Side- chain  theory,  first  application  to  haemolysins 5 

exposition  of 372 

Snake  venom  (see  also  Cobra  venom) /. . .  291 

lecithids  of 466 

studies  on 291 

Soaps,  haemolytic  action  of 464 

Specificity,  limitation  of  term 100,  242 

of  amboceptors 584 

of  immune  sera,  nature  of 50 

use  of  term  in  immunity 561 

Specific  therapeutics 404 

Spectrum,  of  diphtheria  toxin 490,  493,  552 

Spermatozoa,  immune  serum  against 24 

Spermatoxin 48,  52,  193 

production  of,  in  castrated  rabbits 48 

Spider,  poison  of 167 

poisoning  by 173 

Spleen,  as  source  of  immune  bodies 5 

complement  content  of 44 

Staining,  analogy  to  binding  of  lysins 75 

Standing,  effect  of,  on  determinations  of  Lf 669 

Staphylotoxin,  toxoid  of 82 

Stereochemical  conception,  of  complement-immune  body  combination 63 

Stroma,  of  blood-cells,  in  anchoring  immune  body 74 

Stromata,  of  blood-cells,  mode  of  preparation f 171 

Substance  sensibilitrice 57,  381,  469,  562 

Surface  attraction,  in  absorption  of  complement 200 

lack  of  specificity 78 

Tartar  emetic,  as  trypanocidal  agent 687 

Teleological  significance,  of  amboceptor  action 563 

Temperature,  use  of  low,  for  combining  experiments 6,  12 

Tetanolysin,  cholesterin  in  relation  to  haemolysis  by 455 

Tetanus  antitoxin,  effect  of,  plus  brain 60 

in  frogs .  .  ... 91 

Tetanus  toxin,  combination  with  nerve  tissue 77 

neutralization  by  brain 356 

union  with  brain  tissue 5 

Therapeutics,  specific 404 

Thermolabile,  definition  of  the  term 340 

Thermostable,  definition  of  the  term 340 

Tissue  cells,  complexity  of  their  side-chains 43 

Tissue  receptors,  affinity  of 163 

Titoxin 556 

Toad,  toxin  of  toads 175 

Toluol,  as  preservative 176 

Toxin,  composed  of  two  groups 57 


712  INDEX   OF   SUBJECTS 

PAGE 

Toxin,  decomposition  of 486 

neutralization  of,  by  antitoxin 369 

of  toads 176 

recovery  of,  from  toxin-antitoxin  combinations 672 

spectrum  of,  so-called 490,  493,  552 

supposed  transformation  into  antitoxin 366 

various  kinds  of 391 

Toxin-antitoxin,  combination  in  varying  proportions 512 

dissociation  of 666 

study  of  the  reaction 514,  547 

Toxin-toxoid,  an  irreversible  reaction 502 

Toxinan 556 

Toxoid  changes,  in  various  toxins 517 

Toxoids,  definition  of 369 

influence  on  toxin-antitoxin  reaction 488 

nature  of 80 

various  kinds  of 492 

Toxons 503,  507 

existence  of,  demonstrated 577 

Toxonoid 506 

Tracheal  epithelium,  immune  serum  against 38,  49 

Triceptor 112 

Trypanocidal  substances 687 

Trypanosomes,  Ehrlich's  studies  on 687 

serum-fast  varieties 684 

Trypan  red  (trypanrot) 687 

Triphenylmethane  dyes,  as  trypanocidal  agents 687 

Tritoxoid 495 

Typhoid  bacillus,  action  of  normal  sera  on 589 

Typhoid  immune  bodies,  source  of 5 

Urine,  immunizing  with,  to  produce  hsemolysins 244 

Vibrio  choleras,  bacteriolysis  of 1 

Vibrio  Metchnikoff 133,  122 

Vibrio  Nordhafen 124 

Weigert's  theory,  of  super-regeneration 373 

Yeast  cells,  to  absorb  complement 42,  213 

k 

Zwischenkorper  (see  Interbody). 
Zymotoxic  group,  of  complements 65 


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